Secreted and transmembrane polypeptides and nucleic acids encoding the same

ABSTRACT

The present invention is directed to novel polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

RELATED APPLICATIONS

This application is a continuation of, and claims priority under 35 USC§120 to, U.S. application Ser. No. 09/866,034 filed May 25, 2001, whichis a continuation of, and claims priority under 35 USC §120 to, PCTApplication PCT/US99/28634 filed Dec. 1, 1999, which claims priorityunder 35 USC §119 to U.S. Provisional Application No. 60/119,965 filedFeb. 12, 1999.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of novel DNA and to the recombinant production of novelpolypeptides.

BACKGROUND OF THE INVENTION

Extracellular proteins play important roles in, among other things, theformation, differentiation and maintenance of multicellular organisms.The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiationfactors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment.

Secreted proteins have various industrial applications, including aspharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents. Efforts are being undertaken by both industry and academia toidentify new, native secreted proteins. Many efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel secreted proteins. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci. 93:7108-7113 (1996); U.S. Pat. No.5,536,637)].

Membrane-bound proteins and receptors can play important roles in, amongother things, the formation, differentiation and maintenance ofmulticellular organisms. The fate of many individual cells, e.g.,proliferation, migration, differentiation, or interaction with othercells, is typically governed by information received from other cellsand/or the immediate environment. This information is often transmittedby secreted polypeptides (for instance, mitogenic factors, survivalfactors, cytotoxic factors, differentiation factors, neuropeptides, andhormones) which are, in turn, received and interpreted by diverse cellreceptors or membrane-bound proteins. Such membrane-bound proteins andcell receptors include, but are not limited to, cytokine receptors,receptor kinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interactions. The membrane-boundproteins can also be employed for screening of potential peptide orsmall molecule inhibitors of the relevant receptor/ligand interaction.

Efforts are being undertaken by both industry and academia to identifynew, native receptor or membrane-bound proteins. Many efforts arefocused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel receptor or membrane-boundproteins.

1. PRO1800

Hep27 protein is synthesized and accumulated in the nucleus of humanhepatoblastoma cells (HepG2 cells) following growth arrest induced bybutyrate treatment (Gabrielli et al., Eur. J. Biochem. 232:473-477(1995)). The synthesis of Hep27 is inhibited in cells that, releasedfrom the butyrate block, have resumed DNA synthesis. The Hep27 proteinsequence shows significant homology to the known short-chain alcoholdehydrogenase (SCAD) family of proteins and it has been suggested thatHep27 is a new member of the SCAD family of proteins. In agreement withits nuclear localization, Hep27 has a region similar to the bipartitenuclear-targeting sequence and Hep27 mRNA expression and proteinsynthesis suggests the existence of a regulation at thepost-transcriptional level.

We herein describe the identification and characterization of novelpolypeptides having homology to Hep27 protein, designated herein asPRO1800 polypeptides.

2. PRO539

Development of multicellular organisms depends, at least in part, onmechanisms which specify, direct or maintain positional information topattern cells, tissues, or organs. Various secreted signaling molecules,such as members of the transforming growth factor-beta (TGF-β), Wnt,fibroblast growth factors and hedgehog families have been associatedwith patterning activity of different cells and structures in Drosophilaas well as in vertebrates. Perrimon, Cell 80:517-520 (1995).

Costal-2 is a novel kinesin-related protein in the Hedgehog signalingpathway. Hedgehog (Hh) was first identified as a segment-polarity geneby a genetic screen in Drosophila melanogaster, Nusslein-Volhard et al.Roux. Arch. Dev. Biol. 193: 267-282 (1984), that plays a wide variety ofdevelopmental functions. Perrimon, supra. Although only one DrosophilaHh gene has been identified, three mammalian Hh homologues have beenisolated: Sonic Hh (SHh), Desert Hh (DHh) and Indian Hh (IHh), Echelardet al., Cell 75: 1417-30 (1993); Riddle et al., Cell 75: 1401-16 (1993).SHh is expressed at high level in the notochord and floor plate ofdeveloping vertebrate embryos. In vitro explant assays as well asectopic expression of SHh in transgenic animals show that SHh plays akey role in neuronal tube patterning, Echelard et al., supra., Krauss etal., Cell 75, 1432-44 (1993), Riddle et al., Cell 75: 1401-16 (1993),Roelink et al, Cell 81: 445-55 (1995). In vitro explant assays as wellas ectopic expression of SHh in transgenic animals show that SHh plays akey role in neural tube patterning, Echelard et al. (1993), supra.;Ericson et al., Cell 81: 747-56 (1995); Marti et al., Nature 375: 322-5(1995); Roelink et al. (1995), supra; Hynes et al., Neuron 19: 15-26(1997). Hh also plays a role in the development of limbs (Krauss et al.,Cell 75: 1431-44 (1993); Laufer et al., Cell 79, 993-1003 (1994)),somites (Fan and Tessier-Lavigne, Cell 79, 1175-86 (1994); Johnson etal., Cell 79: 1165-73 (1994)), lungs (Bellusci et al., Develop. 124:53-63 (1997) and skin (Oro et al., Science 276: 817-21 (1997). Likewise,IHh and DHh are involved in bone, gut and germinal cell development,Apelqvist et al., Curr. Biol. 7: 801-4 (1997); Bellusci et al., Dev.Suppl. 124: 53-63 (1997); Bitgood et al., Curr. Biol. 6: 298-304 (1996);Roberts et al., Development 121: 3163-74 (1995). SHh knockout micefurther strengthened the notion that SHh is critical to many aspect ofvertebrate development, Chiang et al., Nature 383: 407-13 (1996). Thesemice show defects in midline structures such as the notochord and thefloor plate, absence of ventral cell types in neural tube, absence ofdistal limb structures, cyclopia, and absence of the spinal column andmost of the ribs.

At the cell surface, the Hh signals is thought to be relayed by the 12transmembrane domain protein Patched (Ptch) [Hooper and Scott, Cell 59:751-65 (1989); Nakano et al., Nature 341: 508-13 (1989)] and theG-protein coupled like receptor Smoothened (Smo) [Alcedo et al., Cell86: 221-232 (1996); van den Heuvel and Ingham, Nature 382: 547-551(1996)]. Both genetic and biochemical evidence support a receptor modelwhere Ptch and Smo are part of a multicomponent receptor complex, Chenand Struhl, Cell 87: 553-63 (1996); Marigo et al., Nature 384: 176-9(1996); Stone et al., Nature 384: 129-34 (1996). Upon binding of Hh toPtch, the normal inhibitory effect of Ptch on Smo is relieved, allowingSmo to transduce the Hh signal across the plasma membrane. Loss offunction mutations in the Ptch gene have been identified in patientswith the basal cell nevus syndrome (BCNS), a hereditary diseasecharacterized by multiple basal cell carcinomas (BCCs). DisfunctionalPtch gene mutations have also been associated with a large percentage ofsporadic basal cell carcinoma tumors, Chidambaram et al., CancerResearch 56: 4599-601 (1996); Gailani et al., Nature Genet. 14: 78-81(1996); Hahn et al., Cell 85: 841-51 (1996); Johnson et al., Science272: 1668-71 (1996); Unden et al., Cancer Res. 56: 4562-5; Wicking etal., Am. J. Hum. Genet. 60: 21-6 (1997). Loss of Ptch function isthought to cause an uncontrolled Smo signaling in basal cell carcinoma.Similarly, activating Smo mutations have been identified in sporatic BCCtumors (Xie et al., Nature 391: 90-2 (1998)), emphasizing the role ofSmo as the signaling subunit in the receptor complex for SHh. However,the exact mechanism by which Ptch controls Smo activity still has yet tobe clarified and the signaling mechanisms by which the Hh signal istransmitted from the receptor to downstream targets also remain to beelucidated. Genetic epistatic analysis in Drosophila has identifiedseveral segment-polarity genes which appear to function as components ofthe Hh signal transduction pathway, Ingham, Curr. Opin. Genet. Dev. 5:492-8 (1995); Perrimon, supra. These include a kinesin-like molecule,Costal-2 (Cos-2) [Robbins et al., Cell 90: 225-34 (1997); Sisson et al.,Cell 90: 235-45 (1997)], a protein designated fused [Preat et al.,Genetics 135: 1047-62 (1990); Therond et al., Proc. Natl. Acad Sci. USA93: 4224-8 (1996)], a novel molecule with unknown function designatedSuppressor of fused [Pham et al., Genetics 140: 587-98 (1995); Preat,Genetics 132: 725-36 (1992)] and a zinc finger protein Ci. [Alexandre etal., Genes Dev. 10: 2003-13 (1996); Dominguez et al., Science 272:1621-5 (1996); Orenic et al, Genes Dev. 4: 1053-67 (1990)]. Additionalelements implicated in Hh signaling include the transcription factor CBP[Akimaru et al., Nature 386: 735-738 (1997)], the negative regulatorslimb [Jiang and Struhl, Nature 391: 493-496 (1998)] and the SHhresponse element COUP-TFII [Krishnan et al., Science 278: 1947-1950(1997)].

Mutants in Cos-2 are embryonicly lethal and display a phenotype similarto Hh over expression, including duplications of the central componentof each segment and expansion domain of Hh responsive genes. Incontrast, mutant embryos for fused and Ci show a phenotype similar to Hhloss of function including deletion of the posterior part of eachsegment and replacement of a mirror-like image duplication of theanterior part or each segment and replacement of a mirror-likeduplication of the anterior part, Busson et al., Roux. Arch. Dev. Biol.197: 221-230 (1988). Molecular characterizations of Ci suggested that itis a transcription factor which directly activates Hh responsive genessuch as Wingless and Dpp, Alexandre et al., (1996) supra, Dominguez etal., (1996) supra. Likewise, molecular analysis of fused reveals that itis structurally related to serine threonine kinases and that both intactN-terminal kinase domain and a C-terminal regulatory region are requiredfor its proper function, Preat et al., Nature 347: 87-9 (1990); Robbinset al., (1997), supra; Therond et al., Proc. Natl. Acad. Sci. USA 93:4224-8 (1996). Consistent with the putative opposing functions of Cos-2and fused, fused mutations are suppressed by Cos-2 mutants and also bySuppressor of fused mutants, Preat et al., Genetics 135: 1047-62 (1993).However, whereas fused null mutations and N-terminal kinase domainmutations can be fully suppressed by Suppressor of fused mutations,C-terminus mutations of fused display a strong Cos-2 phenotype in aSuppressor of fused background. This suggests that the fused kinasedomain can act as a constitutive activator of SHh signaling whenSuppressor of Fused is not present. Recent studies have shown that the92 kDa Drosophila fused, Cos-2 and Ci are present in a microtubuleassociated multiprotein complex and that Hh signaling leads todissociation of this complex from microtubules, Robbins et al, Cell 90:225-34 (1997); Sisson et al., Cell 90: 235-45 (1997). Both fused andCos-2 become phosphorylated in response to Hh treatment, Robbins et al.,supra; Therond et al., Genetics 142: 1181-98 (1996), but the kinase(s)responsible for this activity(ies) remain to be characterized. To date,the only known vertebrate homologues for these components are members ofthe Gli protein family (e.g., Gli-1, Gli-2 and Gli-3). These are zincfinger putative transcription factors that are structurally related toCi. Among these, Gli-1 was shown to be a candidate mediator of the SHhsignal [Hynes et al., Neuron 15: 35-44 (1995), Lee et al., Development124: 2537-52 (1997); Alexandre et al., Genes Dev. 10: 2003-13 (1996)]suggesting that the mechanism of gene activation in response to Hh maybe conserved between fly and vertebrates. To determine whether othersignaling components in the Hh cascade are evolutionarily conserved andto examine the function of fused in the Hh signaling cascade on thebiochemical level, Applicants have isolated and characterized the humanfused cDNA. Tissue distribution on the mouse indicates that fused isexpressed in SHh responsive tissues. Biochemical studies demonstratethat fused is a functional kinase. Functional studies provide evidencethat fused is an activator of Gli and that a dominant negative form offused is capable of blocking SHh signaling in Xenopus embryos. Togetherthis data demonstrated that both Cos-2 and fused are directly involvedin Hh signaling.

For additional references related to the Costal-2 protein, see Simpsonet al., Dev. Biol. 122:201-209 (1987), Grau et al., Dev. Biol.122:186-200 (1987), Preat et al., Genetics 135:1047-1062 (1993), Sissonet al., Cell 90:235-245 (1997) and Robbins et al., Cell 90:225-234(1997).

Applicants have herein identified and describe a cDNA encoding a humanCostal-2 homolog polypeptide, designated herein as PRO539.

3. PRO982

Efforts are being undertaken by both industry and academia to identifynew, native secreted proteins. Many efforts are focused on the screeningof mammalian recombinant DNA libraries to identify the coding sequencesfor novel secreted proteins. We herein describe the identification andcharacterization of novel secreted polypeptides, designated herein asPRO982 polypeptides.

4. PRO1434

The nel gene has been described to encode a protein that is expressed inthe neural tissues of chicken (Watanabe et al., Genomics 38(3):273-276(1996)). Recently, two novel human cDNAs (designated NELL1 and NELL2)have been isolated and characterized which encode polypeptides havinghomology to that encoded by the chicken nel gene, wherein those humanpolypeptides contain six EGF-like repeats (Watanabe et al., supra).Given the neural-specific expression of these genes, it is suggestedthat they may play a role in neural development. There is, therefore,significant interest in identifying and characterizing novelpolypeptides having homology to net, NELL1 and NELL2.

We herein describe the identification and characterization of novelpolypeptides having homology to the net protein, designated herein asPRO1434 polypeptides.

5. PRO1863

Efforts are being undertaken by both industry and academia to identifynew, native transmembrane proteins. Many efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel transmembrane proteins. We herein describe theidentification and characterization of novel transmembrane polypeptides,designated herein as PRO1863 polypeptides.

6. PRO1917

The characterization of inositol phosphatases is of interest because itis fundamental to the understanding of signaling activities thatstimulate the release of Ca²⁺ from the endoplasmic reticulum. Molecularcloning allowed the identification of a multiple inositol polyphosphatephosphatase which is highly expressed in kidney and liver (Craxton etal. (1997) Biochem J. 328:75-81).

7. PRO1868

The inflammatory response is complex and is mediated by a variety ofsignaling molecules produced locally by mast cells, nerve endings,platelets, leucocytes and complement activation. Certain of thesesignaling molecules cause the endothelial cell lining to become moreporous and/or even to express selectins which act as cell surfacemolecules which recognize and attract leucocytes through specificcarbohydrate recognition. Stronger leucocyte binding is mediated byintegrins, which mediate leukocyte movement through the endothelium.Additional signaling molecules act as chemoattractants, causing thebound leucocytes to crawl towards the source of the attractant. Othersignaling molecules produced in the course of an inflammatory responseescape into the blood and stimulate the bone marrow to produce moreleucocytes and release them into the blood stream.

Inflammation is typically initiated by an antigen, which can bevirtually any molecule capable of initiating an immune response. Undernormal physiological conditions these are foreign molecules, butmolecules generated by the organism itself can serve as the catalyst asis known to occur in various disease states.

T-cell proliferation is a mixed lymphocyte culture or mixed lymphocytereaction (MLR) is an established indication of the ability of a compoundto stimulate the immune system. In an inflammatory response, theresponding leucocytes can be neutrophilic, eosinophilic, monocytic orlymphocytic. Histological examination of the affected tissues providesevidence of an immune stimulating or inhibiting response. See CurrentProtocols in Immunology, ed. John E. Coligan, 1994, John Wiley and Sons,Inc.

Inflammatory bowel disease (IBD) is a term used to collectively describegut disorders including both ulcerative colitis (UC) and Crohn'sdisease, both of which are classified as distinct disorders, but sharecommon features and likely share pathology. The commonality of thediagnostic criteria can make it difficult to precisely determine whichof the two disorders a patient has; however the type and location of thelesion in each are typically different. UC lesions arecharacteristically a superficial ulcer of the mucosa and appear in thecolon, proximal to the rectum. CD lesions are characteristicallyextensive linear fissures, and can appear anywhere in the bowel,occasionally involving the stomach, esophagus and duodenum.

Conventional treatments for IBD usually involve the administration ofantiinflammatory or immunosuppressive agents, such as sulfasalazine,corticosteriods, 6-mercaptopurine/azathoprine, or cyclospoine all ofwhich only bring partial relief to the afflicted patient. However, whenantiinflammatory/immunosuppressive therapies fail, colectomies are thelast line of defense. Surgery is required for about 30% of CD patientswithin the first year after diagnosis, with the likelihood for operativeprocedure increasing about 5% annually thereafter. Unfortunately, CDalso has a high rate of reoccurrence as about 5% of patients requiresubsequent surgery after the initial year. UC patients further have asubstantially increased risk of developing colorectal cancer.Presumably, this is due to the recurrent cycles of injury to theepithelium, followed by regrowth, which continually increases the riskof neoplastic transformation.

A recently discovered member of the immunoglobulin superfamily known asJunctional Adhesion Molecule (JAM) has been identified to be selectivelyconcentrated at intercellular junctions of endothelial and epithelialcells of different origins. Martin-Padura, I. et al., J. Cell Biol.142(1): 117-27 (1998). JAM is a type I integral membrane protein withtwo extracellular, intrachain disulfide loops of the V-type. JAM bearssubstantial homology to A33 antigen (FIG. 1 or FIG. 18). A monoclonalantibody directed to JAM was found to inhibit spontaneous andchemokine-induced monocyte transmigration through an endothelial cellmonolayer in vitro. Martin-Padura, supra.

It has been recently discovered that JAM expression is increased in thecolon of CRF2-4−/− mice with colitis. CRF 2-4−/− (IL-10R subunitknockout mice) develop a spontaneous colitis mediated by lymphocytes,monocytes and neutrophils. Several of the animals also developed colonadenocarcinoma. As a result, it is foreseeable likely that the compoundsof the invention are expressed in elevated levels in or otherwiseassociated with human diseases such as inflammatory bowel disease, otherinflammatory diseases of the gut as well as colorectal carcinoma.

The compounds of the invention also bear significant homology to A33antigen, a known colorectal cancer-associated marker. The A33 antigen isexpressed in more than 90% of primary or metastatic colon cancers aswell as normal colon epithelium. In carcinomas originating from thecolonic mucosa, the A33 antigen is expressed homogeneously in more than95% of all cases. The A33 antigen, however, has not been detected in awide range of other normal issues, i.e., its expression appears to beorgan specific. Therefore, the A33 antigen appears to play an importantrole in the induction of colorectal cancer.

Since colon cancer is a widespread disease, early diagnosis andtreatment is an important medical goal. Diagnosis and treatment of coloncancer can be implemented using monoclonal antibodies (mAbs) specifictherefore having fluorescent, nuclear magnetic or radioactive tags.Radioactive gene, toxins and/or drug tagged mAbs can be used fortreatment in situ with minimal patient description. mAbs can also beused to diagnose during the diagnosis and treatment of colon cancers.For example, when the serum levels of the A33 antigen are elevated in apatient, a drop of the levels after surgery would indicate the tumorresection was successful. On the other hand, a subsequent rise in serumA33 antigen levels after surgery would indicate that metastases of theoriginal tumor may have formed or that new primary tumors may haveappeared.

Such monoclonal antibodies can be used in lieu of, or in conjunctionwith surgery and/or other chemotherapies. For example, preclinicalanalysis and localization studies in patients infected with colorectalcarcinoma with a mAb to A33 are described in Welt et al., J. Clin.Oncol. 8: 1894-1906 (1990) and Welt et al., J. Clin. Oncol. 12:1561-1571 (1994), while U.S. Pat. No. 4,579,827 and U.S. Ser. No.424,991 (E.P. 199,141) are directed to the therapeutic administration ofmonoclonal antibodies, the latter of which relates to the application ofanti-A33 mAb.

We herein describe the identification and characterization of novelpolypeptides having homology to A33 antigen protein, designated hereinas PRO1868 polypeptides.

8. PRO3434

Efforts are being undertaken by both industry and academia to identifynew, native secreted proteins. Many efforts are focused on the screeningof mammalian recombinant DNA libraries to identify the coding sequencesfor novel secreted proteins. We herein describe the identification andcharacterization of novel secreted polypeptides, designated herein asPRO3434 polypeptides.

9. PRO1927

Proteins are glycosylated by a complex set of reactions which aremediated by membrane bound glycosyltransferases. There is a large numberof different glycosyltransferases that account for the array ofcarbohydrate structures synthesized. N-acetylglucosaminyltransferaseproteins comprise a family of glycosyltransferases that provide for avariety of important biological functions in the mammalian organism. Asan example, UDP-N-acetylglucosamine: alpha-3-D-mannosidebeta-1,2-N-acetylglucosaminyltransferase I is an glycosyltransferasethat catalyzes an essential first step in the conversion of high-mannoseN-glycans to hybrid and complex N-glycans (Sarkar et al., Proc. Natl.Acad. Sci. USA. 88:234-238 (1991). UPD-N-acetylglucosamine: alpha1,3-D-mannoside beta 1,4-N-acetylglucosaminyltransferase is an essentialenzyme in the production of tri- and tetra-antennary asparagine-linkedsugar chains, and has been recently been purified from bovine smallintestine using cDNA cloning (Minowa et al., J. Biol. Chem. (1998)273(19): 11556-62). There is interest in the identification andcharacterization of additional members of theN-acetylglucosaminyltransferase protein family, and more generally, theidentification of novel glycosyltransferases.

SUMMARY OF THE INVENTION

1. PRO1800

A cDNA clone (DNA35672-2508) has been identified, having homology tonucleic acid encoding Hep27 protein, that encodes a novel polypeptide,designated in the present application as “PRO1800”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1800 polypeptide.

In one aspect, the isolated nucleic acid comprises DNA having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to (a) a DNA moleculeencoding a PRO1800 polypeptide having the sequence of amino acidresidues from about 1 or about 16 to about 278, inclusive of FIG. 2 (SEQID NO:2), or (b) the complement of the DNA molecule of (a).

In another aspect, the invention concerns an isolated nucleic acidmolecule encoding a PRO1800 polypeptide comprising DNA hybridizing tothe complement of the nucleic acid between about nucleotides 36 or about81 and about 869, inclusive, of FIG. 1 (SEQ ID NO:1). Preferably,hybridization occurs under stringent hybridization and wash conditions.

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA having at least about 80% sequence identity,preferably at least about 85% sequence identity, more preferably atleast about 90% sequence identity, most preferably at least about 95%sequence identity to (a) a DNA molecule encoding the same maturepolypeptide encoded by the human protein cDNA in ATCC Deposit No. 203538(DNA35672-2508) or (b) the complement of the nucleic acid molecule of(a). In a preferred embodiment, the nucleic acid comprises a DNAencoding the same mature polypeptide encoded by the human protein cDNAin ATCC Deposit No. 203538 (DNA35672-2508).

In still a further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to the sequence of aminoacid residues 1 or about 16 to about 278, inclusive of FIG. 2 (SEQ IDNO:2), or (b) the complement of the DNA of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule having at least 230 nucleotides and produced by hybridizing atest DNA molecule under stringent conditions with (a) a DNA moleculeencoding a PRO1800 polypeptide having the sequence of amino acidresidues from 1 or about 16 to about 278, inclusive of FIG. 2 (SEQ IDNO:2), or (b) the complement of the DNA molecule of (a), and, if the DNAmolecule has at least about an 80% sequence identity, prefereably atleast about an 85% sequence identity, more preferably at least about a90% sequence identity, most preferably at least about a 95% sequenceidentity to (a) or (b), isolating the test DNA molecule.

In a specific aspect, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1800 polypeptide, with or withoutthe N-terminal signal sequence and/or the initiating methionine, or iscomplementary to such encoding nucleic acid molecule. The signal peptidehas been tentatively identified as extending from about amino acidposition 1 to about amino acid position 15 in the sequence of FIG. 2(SEQ ID NO:2).

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1or about 16 to about 278, inclusive of FIG. 2 (SEQ ID NO:2), or (b) thecomplement of the DNA of (a).

Another embodiment is directed to fragments of a PRO1800 polypeptidecoding sequence that may find use as hybridization probes. Such nucleicacid fragments may be from about 20 to about 80 nucleotides in length,preferably from about 20 to about 60 nucleotides in length, morepreferably from about 20 to about 50 nucleotides in length and mostpreferably from about 20 to about 40 nucleotides in length and may bederived from the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1).

In another embodiment, the invention provides isolated PRO1800polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

In a specific aspect, the invention provides isolated native sequencePRO1800 polypeptide, which in certain embodiments, includes an aminoacid sequence comprising residues 1 or about 16 to about 278 of FIG. 2(SEQ ID NO:2).

In another aspect, the invention concerns an isolated PRO1800polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 or about 16 to about 278, inclusive of FIG. 2 (SEQ ID NO:2).

In a further aspect, the invention concerns an isolated PRO1800polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 orabout 16 to about 278, inclusive of FIG. 2 (SEQ ID NO:2).

In yet another aspect, the invention concerns an isolated PRO1800polypeptide, comprising the sequence of amino acid residues 1 or about16 to about 278, inclusive of FIG. 2 (SEQ ID NO:2), or a fragmentthereof sufficient to provide a binding site for an anti-PRO1800antibody. Preferably, the PRO1800 fragment retains a qualitativebiological activity of a native PRO1800 polypeptide.

In a still further aspect, the invention provides a polypeptide producedby (i) hybridizing a test DNA molecule under stringent conditions with(a) a DNA molecule encoding a PRO1800 polypeptide having the sequence ofamino acid residues from about 1 or about 16 to about 278, inclusive ofFIG. 2 (SEQ ID NO: 2), or (b) the complement of the DNA molecule of (a),and if the test DNA molecule has at least about an 80% sequenceidentity, preferably at least about an 85% sequence identity, morepreferably at least about a 90% sequence identity, most preferably atleast about a 95% sequence identity to (a) or (b), (ii) culturing a hostcell comprising the test DNA molecule under conditions suitable forexpression of the polypeptide, and (iii) recovering the polypeptide fromthe cell culture.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO1800 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-PRO1800 antibody.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists of a native PRO1800 polypeptide by contactingthe native PRO1800 polypeptide with a candidate molecule and monitoringa biological activity mediated by said polypeptide.

In a still further embodiment, the invention concerns a compositioncomprising a PRO1800 polypeptide, or an agonist or antagonist ashereinabove defined, in combination with a pharmaceutically acceptablecarrier.

2. PRO539

A cDNA clone (DNA47465-1561) has been identified, having homology tonucleic acid encoding Costal-2 protein, that encodes a novelpolypeptide, designated in the present application as “PRO539”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO539 polypeptide.

In one aspect, the isolated nucleic acid comprises DNA having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to (a) a DNA moleculeencoding a PRO539 polypeptide having the sequence of amino acid residuesfrom about 1 to about 830, inclusive of FIG. 4 (SEQ ID NO:7), or (b) thecomplement of the DNA molecule of (a).

In another aspect, the invention concerns an isolated nucleic acidmolecule encoding a PRO539 polypeptide comprising DNA hybridizing to thecomplement of the nucleic acid between about nucleotides 186 and about2675, inclusive, of FIG. 3 (SEQ ID NO:6). Preferably, hybridizationoccurs under stringent hybridization and wash conditions.

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA having at least about 80% sequence identity,preferably at least about 85% sequence identity, more preferably atleast about 90% sequence identity, most preferably at least about 95%sequence identity to (a) a DNA molecule encoding the same maturepolypeptide encoded by the human protein cDNA in ATCC Deposit No. 203661(DNA47465-1561) or (b) the complement of the nucleic acid molecule of(a). In a preferred embodiment, the nucleic acid comprises a DNAencoding the same mature polypeptide encoded by the human protein cDNAin ATCC Deposit No. 203661 (DNA47465-1561).

In still a further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to the sequence of aminoacid residues 1 to about 830, inclusive of FIG. 4 (SEQ ID NO:7), or (b)the complement of the DNA of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule having at least 100 nucleotides and produced by hybridizing atest DNA molecule under stringent conditions with (a) a DNA moleculeencoding a PRO539 polypeptide having the sequence of amino acid residuesfrom 1 to about 830, inclusive of FIG. 4 (SEQ ID NO:7), or (b) thecomplement of the DNA molecule of (a), and, if the DNA molecule has atleast about an 80% sequence identity, prefereably at least about an 85%sequence identity, more preferably at least about a 90% sequenceidentity, most preferably at least about a 95% sequence identity to (a)or (b), isolating the test DNA molecule.

In a specific aspect, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO539 polypeptide, with or withoutthe initiating methionine, or is complementary to such encoding nucleicacid molecule.

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1to about 830, inclusive of FIG. 4 (SEQ ID NO:7), or (b) the complementof the DNA of (a).

Another embodiment is directed to fragments of a PRO539 polypeptidecoding sequence that may find use as hybridization probes. Such nucleicacid fragments are from about 20 to about 80 nucleotides in length,preferably from about 20 to about 60 nucleotides in length, morepreferably from about 20 to about 50 nucleotides in length and mostpreferably from about 20 to about 40 nucleotides in length and may bederived from the nucleotide sequence shown in FIG. 3 (SEQ ID NO:6).

In another embodiment, the invention provides isolated PRO539polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

In a specific aspect, the invention provides isolated native sequencePRO539 polypeptide, which in certain embodiments, includes an amino acidsequence comprising residues 1 to about 830 of FIG. 4 (SEQ ID NO:7).

In another aspect, the invention concerns an isolated PRO539polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 to about 830, inclusive of FIG. 4 (SEQ ID NO:7).

In a further aspect, the invention concerns an isolated PRO539polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 toabout 830, inclusive of FIG. 4 (SEQ ID NO:7).

In yet another aspect, the invention concerns an isolated PRO539polypeptide, comprising the sequence of amino acid residues 1 to about830, inclusive of FIG. 4 (SEQ ID NO:7), or a fragment thereof sufficientto provide a binding site for an anti-PRO539 antibody. Preferably, thePRO539 fragment retains a qualitative biological activity of a nativePRO539 polypeptide.

In a still further aspect, the invention provides a polypeptide producedby (i) hybridizing a test DNA molecule under stringent conditions with(a) a DNA molecule encoding a PRO539 polypeptide having the sequence ofamino acid residues from about 1 to about 830, inclusive of FIG. 4 (SEQID NO:7), or (b) the complement of the DNA molecule of (a), and if thetest DNA molecule has at least about an 80% sequence identity,preferably at least about an 85% sequence identity, more preferably atleast about a 90% sequence identity, most preferably at least about a95% sequence identity to (a) or (b), (ii) culturing a host cellcomprising the test DNA molecule under conditions suitable forexpression of the polypeptide, and (iii) recovering the polypeptide fromthe cell culture.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO539 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-PRO539 antibody.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists of a native PRO539 polypeptide by contacting thenative PRO539 polypeptide with a candidate molecule and monitoring abiological activity mediated by said polypeptide. In a preferredembodiment, the biological activity is either binding to microtubiles orthe ability to complex with fused and cubitus interruptus.

In a still further embodiment, the invention concerns a compositioncomprising a PRO539 polypeptide, or an agonist or antagonist ashereinabove defined, in combination with a pharmaceutically acceptablecarrier.

In yet another embodiment, the invention provides for compounds andmethods for developing antagonists against and agonist promoting PRO539modulation of Hedgehog signaling. In particular, an antagonist ofvertebrate PRO539 which blocks, prevents, inhibits and/or neutralizedthe normal functioning of PRO539 in SH signaling pathway, including bothsmall bioorganic molecules and antisense nucleotides.

In yet another embodiment, the invention provides for alternativelyspliced variants of human PRO539.

In still yet a further embodiment, the invention provides a method ofscreening or assaying for identifying molecules that alter the PRO539modulation of hedgehog signaling. Preferably, the molecules eitherprevent interaction of PRO539 with its associative complexing proteins(such as fused or cubitus interruptus) or prevent or inhibitdissociation of complexes. The assay comprises the incubation of amixture comprising PRO539 and a substrate with a candidate molecule anddetection of the ability of the candidate molecule to modulate PRO539hedgehog signaling. The screened molecules preferably are small moleculedrug candidates.

In yet another embodiment, the method relates to a technique ofdiagnosing to determine whether a particular disorder is modulated byhedgehog signaling, comprising:

-   -   (a) culturing test cells or tissues;    -   (b) administering a compound which can inhibit PRO539 modulated        hedgehog signaling; and    -   (c) determining whether hedgehog signaling is modulated.        3. PRO982

A cDNA clone (DNA57700-1408) has been identified that encodes a novelpolypeptide, designated in the present application as “PRO982.”

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO982 polypeptide.

In one aspect, the isolated nucleic acid comprises DNA having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to (a) a DNA moleculeencoding a PRO982 polypeptide having the sequence of amino acid residuesfrom 1 or about 22 to about 125, inclusive of FIG. 6 (SEQ ID NO:9), or(b) the complement of the DNA molecule of (a).

In another aspect, the invention concerns an isolated nucleic acidmolecule encoding a PRO982 polypeptide comprising DNA hybridizing to thecomplement of the nucleic acid between about residues 89 and about 400,inclusive, of FIG. 5 (SEQ ID NO:8). Preferably, hybridization occursunder stringent hybridization and wash conditions.

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA having at least about 80% sequence identity,preferably at least about 85% sequence identity, more preferably atleast about 90% sequence identity, most preferably at least about 95%sequence identity to (a) a DNA molecule encoding the same maturepolypeptide encoded by the human protein cDNA in ATCC Deposit No. 203583(DNA57700-1408), or (b) the complement of the DNA molecule of (a). In apreferred embodiment, the nucleic acid comprises a DNA encoding the samemature polypeptide encoded by the human protein cDNA in ATCC Deposit No.203583 (DNA57700-1408).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to the sequence of aminoacid residues from 1 or about 22 to about 125, inclusive of FIG. 6 (SEQID NO:9), or the complement of the DNA of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule having at least about 50 nucleotides, and preferably at leastabout 100 nucleotides and produced by hybridizing a test DNA moleculeunder stringent conditions with (a) a DNA molecule encoding a PRO982polypeptide having the sequence of amino acid residues from 1 or about22 to about 125, inclusive of FIG. 6 (SEQ ID NO:9), or (b) thecomplement of the DNA molecule of (a), and, if the DNA molecule has atleast about an 80% sequence identity, preferably at least about an 85%sequence identity, more preferably at least about a 90% sequenceidentity, most preferably at least about a 95% sequence identity to (a)or (b), isolating the test DNA molecule.

In a specific aspect, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO982 polypeptide, with or withoutthe N-terminal signal sequence and/or the initiating methionine, or iscomplementary to such encoding nucleic acid molecule. The signal peptidehas been tentatively identified as extending from amino acid position 1through about amino acid position 21 in the sequence of FIG. 6 (SEQ IDNO:9)

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1or about 22 to about 125, inclusive of FIG. 6 (SEQ ID NO:9), or (b) thecomplement of the DNA of (a).

Another embodiment is directed to fragments of a PRO982 polypeptidecoding sequence that may find use as hybridization probes. Such nucleicacid fragments are from about 20 to about 80 nucleotides in length,preferably from about 20 to about 60 nucleotides in length, morepreferably from about 20 to about 50 nucleotides in length, and mostpreferably from about 20 to about 40 nucleotides in length.

In another embodiment, the invention provides isolated PRO982polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove defined.

In a specific aspect, the invention provides isolated native sequencePRO982 polypeptide, which in one embodiment, includes an amino acidsequence comprising residues 1 or about 22 to 125 of FIG. 6 (SEQ IDNO:9).

In another aspect, the invention concerns an isolated PRO982polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 or about 22 to about 125, inclusive of FIG. 6 (SEQ ID NO:9).

In a further aspect, the invention concerns an isolated PRO982polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 orabout 22 to 125 of FIG. 6 (SEQ ID NO:9).

In yet another aspect, the invention concerns an isolated PRO982polypeptide, comprising the sequence of amino acid residues 1 or about22 to about 125, inclusive of FIG. 6 (SEQ ID NO:9), or a fragmentthereof sufficient to provide a binding site for an anti-PRO982antibody. Preferably, the PRO982 fragment retains a qualitativebiological activity of a native PRO982 polypeptide.

In a still further aspect, the invention provides a polypeptide producedby (i) hybridizing a test DNA molecule under stringent conditions with(a) a DNA molecule encoding a PRO982 polypeptide having the sequence ofamino acid residues from 1 or about 22 to about 125, inclusive of FIG. 6(SEQ ID NO:9), or (b) the complement of the DNA molecule of (a), and ifthe test DNA molecule has at least about an 80% sequence identity,preferably at least about an 85% sequence identity, more preferably atleast about a 90% sequence identity, most preferably at least about a95% sequence identity to (a) or (b), (ii) culturing a host cellcomprising the test DNA molecule under conditions suitable forexpression of the polypeptide, and (iii) recovering the polypeptide fromthe cell culture.

4. PRO1434

A cDNA clone (DNA68818-2536) has been identified, having homology tonucleic acid encoding nel protein, that encodes a novel polypeptide,designated in the present application as “PRO1434”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1434 polypeptide.

In one aspect, the isolated nucleic acid comprises DNA having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to (a) a DNA moleculeencoding a PRO1434 polypeptide having the sequence of amino acidresidues from about 1 or about 28 to about 325, inclusive of FIG. 8 (SEQID NO: 11), or (b) the complement of the DNA molecule of (a).

In another aspect, the invention concerns an isolated nucleic acidmolecule encoding a PRO1434 polypeptide comprising DNA hybridizing tothe complement of the nucleic acid between about nucleotides 581 orabout 662 and about 1555, inclusive, of FIG. 7 (SEQ ID NO:10).Preferably, hybridization occurs under stringent hybridization and washconditions.

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA having at least about 80% sequence identity,preferably at least about 85% sequence identity, more preferably atleast about 90% sequence identity, most preferably at least about 95%sequence identity to (a) a DNA molecule encoding the same maturepolypeptide encoded by the human protein cDNA in ATCC Deposit No. 203657(DNA68818-2536) or (b) the complement of the nucleic acid molecule of(a). In a preferred embodiment, the nucleic acid comprises a DNAencoding the same mature polypeptide encoded by the human protein cDNAin ATCC Deposit No. 203657 (DNA68818-2536).

In still a further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to the sequence of aminoacid residues 1 or about 28 to about 325, inclusive of FIG. 8 (SEQ IDNO: 11), or (b) the complement of the DNA of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule having at least 65 nucleotides and produced by hybridizing atest DNA molecule under stringent conditions with (a) a DNA moleculeencoding a PRO1434 polypeptide having the sequence of amino acidresidues from 1 or about 28 to about 325, inclusive of FIG. 8 (SEQ IDNO:11), or (b) the complement of the DNA molecule of (a), and, if theDNA molecule has at least about an 80% sequence identity, prefereably atleast about an 85% sequence identity, more preferably at least about a90% sequence identity, most preferably at least about a 95% sequenceidentity to (a) or (b), isolating the test DNA molecule.

In a specific aspect, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1434 polypeptide, with or withoutthe N-terminal signal sequence and/or the initiating methionine, and itssoluble, i.e., transmembrane domain deleted or inactivated variants, oris complementary to such encoding nucleic acid molecule. The signalpeptide has been tentatively identified as extending from about aminoacid position 1 to about amino acid position 27 in the sequence of FIG.8 (SEQ ID NO:11). The transmembrane domain has been tentativelyidentified as extending from about amino acid position 11 to about aminoacid position 30 in the PRO1434 amino acid sequence (FIG. 8, SEQ IDNO:11).

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1or about 28 to about 325, inclusive of FIG. 8 (SEQ ID NO:11), or (b) thecomplement of the DNA of (a).

Another embodiment is directed to fragments of a PRO1434 polypeptidecoding sequence that may find use as hybridization probes. Such nucleicacid fragments are from about 20 to about 80 nucleotides in length,preferably from about 20 to about 60 nucleotides in length, morepreferably from about 20 to about 50 nucleotides in length and mostpreferably from about 20 to about 40 nucleotides in length and may bederived from the nucleotide sequence shown in FIG. 7 (SEQ ID NO:10).

In another embodiment, the invention provides isolated PRO1434polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

In a specific aspect, the invention provides isolated native sequencePRO1434 polypeptide, which in certain embodiments, includes an aminoacid sequence comprising residues 1 or about 28 to about 325 of FIG. 8(SEQ ID NO:11).

In another aspect, the invention concerns an isolated PRO1434polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 or about 28 to about 325, inclusive of FIG. 8 (SEQ ID NO:11).

In a further aspect, the invention concerns an isolated PRO1434polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 orabout 28 to about 325, inclusive of FIG. 8 (SEQ ID NO:11).

In yet another aspect, the invention concerns an isolated PRO1434polypeptide, comprising the sequence of amino acid residues 1 or about28 to about 325, inclusive of FIG. 8 (SEQ ID NO:11), or a fragmentthereof sufficient to provide a binding site for an anti-PRO1434antibody. Preferably, the PRO1434 fragment retains a qualitativebiological activity of a native PRO1434 polypeptide.

In a still further aspect, the invention provides a polypeptide producedby (i) hybridizing a test DNA molecule under stringent conditions with(a) a DNA molecule encoding a PRO1434 polypeptide having the sequence ofamino acid residues from about 1 or about 28 to about 325, inclusive ofFIG. 8 (SEQ ID NO:11), or (b) the complement of the DNA molecule of (a),and if the test DNA molecule has at least about an 80% sequenceidentity, preferably at least about an 85% sequence identity, morepreferably at least about a 90% sequence identity, most preferably atleast about a 95% sequence identity to (a) or (b), (ii) culturing a hostcell comprising the test DNA molecule under conditions suitable forexpression of the polypeptide, and (iii) recovering the polypeptide fromthe cell culture.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO1434 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-PRO1434 antibody.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists of a native PRO1434 polypeptide by contactingthe native PRO1434 polypeptide with a candidate molecule and monitoringa biological activity mediated by said polypeptide.

In a still further embodiment, the invention concerns a compositioncomprising a PRO1434 polypeptide, or an agonist or antagonist ashereinabove defined, in combination with a pharmaceutically acceptablecarrier.

5. PRO1863

A cDNA clone (DNA59847-2510) has been identified that encodes a noveltransmembrane polypeptide, designated in the present application as“PRO1863”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1863 polypeptide.

In one aspect, the isolated nucleic acid comprises DNA having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to (a) a DNA moleculeencoding a PRO1863 polypeptide having the sequence of amino acidresidues from about 1 or about 16 to about 437, inclusive of FIG. 10(SEQ ID NO: 16), or (b) the complement of the DNA molecule of (a).

In another aspect, the invention concerns an isolated nucleic acidmolecule encoding a PRO1863 polypeptide comprising DNA hybridizing tothe complement of the nucleic acid between about nucleotides 17 or about62 and about 1327, inclusive, of FIG. 9 (SEQ ID NO:15). Preferably,hybridization occurs under stringent hybridization and wash conditions.

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA having at least about 80% sequence identity,preferably at least about 85% sequence identity, more preferably atleast about 90% sequence identity, most preferably at least about 95%sequence identity to (a) a DNA molecule encoding the same maturepolypeptide encoded by the human protein cDNA in ATCC Deposit No. 203576(DNA59847-2510) or (b) the complement of the nucleic acid molecule of(a). In a preferred embodiment, the nucleic acid comprises a DNAencoding the same mature polypeptide encoded by the human protein cDNAin ATCC Deposit No. 203576 (DNA59847-2510).

In still a further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to the sequence of aminoacid residues 1 or about 16 to about 437, inclusive of FIG. 10 (SEQ IDNO:16), or (b) the complement of the DNA of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule having at least 345 nucleotides and produced by hybridizing atest DNA molecule under stringent conditions with (a) a DNA moleculeencoding a PRO1863 polypeptide having the sequence of amino acidresidues from 1 or about 16 to about 437, inclusive of FIG. 10 (SEQ IDNO:16), or (b) the complement of the DNA molecule of (a), and, if theDNA molecule has at least about an 80% sequence identity, prefereably atleast about an 85% sequence identity, more preferably at least about a90% sequence identity, most preferably at least about a 95% sequenceidentity to (a) or (b), isolating the test DNA molecule.

In a specific aspect, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1863 polypeptide, with or withoutthe N-terminal signal sequence and/or the initiating methionine, and itssoluble, i.e., transmembrane domain deleted or inactivated variants, oris complementary to such encoding nucleic acid molecule. The signalpeptide has been tentatively identified as extending from about aminoacid position 1 to about amino acid position 17 in the sequence of FIG.10 (SEQ ID NO:16). The transmembrane domain has been tentativelyidentified as extending from about amino acid position 243 to aboutamino acid position 260 in the PRO1863 amino acid sequence (FIG. 10, SEQID NO:16).

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1or about 16 to about 437, inclusive of FIG. 10 (SEQ ID NO:16), or (b)the complement of the DNA of (a).

Another embodiment is directed to fragments of a PRO1863 polypeptidecoding sequence that may find use as hybridization probes. Such nucleicacid fragments are from about 20 to about 80 nucleotides in length,preferably from about 20 to about 60 nucleotides in length, morepreferably from about 20 to about 50 nucleotides in length and mostpreferably from about 20 to about 40 nucleotides in length and may bederived from the nucleotide sequence shown in FIG. 9 (SEQ ID NO:15).

In another embodiment, the invention provides isolated PRO1863polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

In a specific aspect, the invention provides isolated native sequencePRO1863 polypeptide, which in certain embodiments, includes an aminoacid sequence comprising residues 1 or about 16 to about 437 of FIG. 10(SEQ ID NO:16).

In another aspect, the invention concerns an isolated PRO1863polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 or about 16 to about 437, inclusive of FIG. 10 (SEQ ID NO:16).

In a further aspect, the invention concerns an isolated PRO1863polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 orabout 16 to about 437, inclusive of FIG. 10 (SEQ ID NO:16).

In yet another aspect, the invention concerns an isolated PRO1863polypeptide, comprising the sequence of amino acid residues 1 or about16 to about 437, inclusive of FIG. 10 (SEQ ID NO:16), or a fragmentthereof sufficient to provide a binding site for an anti-PRO1863antibody. Preferably, the PRO1863 fragment retains a qualitativebiological activity of a native PRO1863 polypeptide.

In a still further aspect, the invention provides a polypeptide producedby (i) hybridizing a test DNA molecule under stringent conditions with(a) a DNA molecule encoding a PRO1863 polypeptide having the sequence ofamino acid residues from about 1 or about 16 to about 437, inclusive ofFIG. 10 (SEQ ID NO:16), or (b) the complement of the DNA molecule of(a), and if the test DNA molecule has at least about an 80% sequenceidentity, preferably at least about an 85% sequence identity, morepreferably at least about a 90% sequence identity, most preferably atleast about a 95% sequence identity to (a) or (b), (ii) culturing a hostcell comprising the test DNA molecule under conditions suitable forexpression of the polypeptide, and (iii) recovering the polypeptide fromthe cell culture.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO1863 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-PRO1863 antibody.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists of a native PRO1863 polypeptide by contactingthe native PRO1863 polypeptide with a candidate molecule and monitoringa biological activity mediated by said polypeptide.

In a still further embodiment, the invention concerns a compositioncomprising a PRO1863 polypeptide, or an agonist or antagonist ashereinabove defined, in combination with a pharmaceutically acceptablecarrier.

6. PRO1917

A cDNA clone (DNA76400-2528) has been identified that encodes a novelpolypeptide having homology to inositol phosphatase and designated inthe present application as “PRO1917”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1917 polypeptide.

In one aspect, the isolated nucleic acid comprises DNA having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to (a) a DNA moleculeencoding a PRO1917 polypeptide having the sequence of amino acidresidues from 1 or about 31 to about 487, inclusive of FIG. 12 (SEQ IDNO:18), or (b) the complement of the DNA molecule of (a).

In another aspect, the invention concerns an isolated nucleic acidmolecule encoding a PRO1917 polypeptide comprising DNA hybridizing tothe complement of the nucleic acid between about residues 96 and about1466, inclusive, of FIG. 11 (SEQ ID NO:17). Preferably, hybridizationoccurs under stringent hybridization and wash conditions.

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA having at least about 80% sequence identity,preferably at least about 85% sequence identity, more preferably atleast about 90% sequence identity, most preferably at least about 95%sequence identity to (a) a DNA molecule encoding the same maturepolypeptide encoded by the human protein cDNA in ATCC Deposit No. 203573(DNA76400-2528), or (b) the complement of the DNA molecule of (a). In apreferred embodiment, the nucleic acid comprises a DNA encoding the samemature polypeptide encoded by the human protein cDNA in ATCC Deposit No.203573 (DNA76400-2528).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to the sequence of aminoacid residues from 1 or about 31 to about 487, inclusive of FIG. 12 (SEQID NO:18), or the complement of the DNA of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule having at least about 50 nucleotides, and preferably at leastabout 100 nucleotides and produced by hybridizing a test DNA moleculeunder stringent conditions with (a) a DNA molecule encoding a PRO1917polypeptide having the sequence of amino acid residues from 1 or about31 to about 487, inclusive of FIG. 12 (SEQ ID NO:18), or (b) thecomplement of the DNA molecule of (a), and, if the DNA molecule has atleast about an 80% sequence identity, preferably at least about an 85%sequence identity, more preferably at least about a 90% sequenceidentity, most preferably at least about a 95% sequence identity to (a)or (b), isolating the test DNA molecule.

In a specific aspect, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1917 polypeptide, with or withoutthe N-terminal signal sequence and/or the initiating methionine, or iscomplementary to such encoding nucleic acid molecule. The signal peptidehas been tentatively identified as extending from amino acid position 1through about amino acid position 30 in the sequence of FIG. 12 (SEQ IDNO:18).

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1or about 31 to about 487, inclusive of FIG. 12 (SEQ ID NO:18), or (b)the complement of the DNA of (a).

Another embodiment is directed to fragments of a PRO1917 polypeptidecoding sequence that may find use as hybridization probes. Such nucleicacid fragments are from about 20 to about 80 nucleotides in length,preferably from about 20 to about 60 nucleotides in length, morepreferably from about 20 to about 50 nucleotides in length, and mostpreferably from about 20 to about 40 nucleotides in length.

In another embodiment, the invention provides isolated PRO1917polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove defined.

In a specific aspect, the invention provides isolated native sequencePRO1917 polypeptide, which in one embodiment, includes an amino acidsequence comprising residues 1 or about 31 to 487 of FIG. 12 (SEQ IDNO:18).

In another aspect, the invention concerns an isolated PRO1917polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 or about 31 to about 487, inclusive of FIG. 12 (SEQ ID NO:18).

In a further aspect, the invention concerns an isolated PRO1917polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 orabout 31 to 487 of FIG. 12 (SEQ ID NO:18).

In yet another aspect, the invention concerns an isolated PRO1917polypeptide, comprising the sequence of amino acid residues 1 or about31 to about 487, inclusive of FIG. 12 (SEQ ID NO:18), or a fragmentthereof sufficient to provide a binding site for an anti-PRO1917antibody. Preferably, the PRO1917 fragment retains a qualitativebiological activity of a native PRO1917 polypeptide.

In a still further aspect, the invention provides a polypeptide producedby (i) hybridizing a test DNA molecule under stringent conditions with(a) a DNA molecule encoding a PRO1917 polypeptide having the sequence ofamino acid residues from 1 or about 31 to about 487, inclusive of FIG.12 (SEQ ID NO:18), or (b) the complement of the DNA molecule of (a), andif the test DNA molecule has at least about an 80% sequence identity,preferably at least about an 85% sequence identity, more preferably atleast about a 90% sequence identity, most preferably at least about a95% sequence identity to (a) or (b), (ii) culturing a host cellcomprising the test DNA molecule under conditions suitable forexpression of the polypeptide, and (iii) recovering the polypeptide fromthe cell culture.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO1917 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-PRO1917 antibody.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists of a native PRO1917 polypeptide, by contactingthe native PRO1917 polypeptide with a candidate molecule and monitoringa biological activity mediated by said polypeptide.

In a still further embodiment, the invention concerns a compositioncomprising a PRO1917 polypeptide, or an agonist or antagonist ashereinabove defined, in combination with a pharmaceutically acceptablecarrier.

7. PRO1868

The present invention concerns compositions and methods for thediagnosis and treatment of inflammatory diseases in mammals, includinghumans. The present invention is based on the identification of proteins(including agonist and antagonist antibodies) which either stimulate orinhibit the immune response in mammals. Inflammatory diseases can betreated by suppressing the inflammatory response. Molecules that enhancean inflammatory response stimulate or potentiate the immune response toan antigen. Molecules which stimulate an inflammatory response can beinhibited where suppression of the inflammatory response would bebeneficial. Molecules which stimulate the inflammatory response can beused therapeutically where enhancement of the inflammatory responsewould be beneficial. Such stimulatory molecules can also be inhibitedwhere suppression of the inflammatory response would be of value.Neutralizing antibodies are examples of molecules that inhibit moleculeshaving immune stimulatory activity and which would be beneficial in thetreatment of inflammatory diseases. Molecules which inhibit theinflammatory response can also be utilized (proteins directly or via theuse of antibody agonists) to inhibit the inflammatory response and thusameliorate inflammatory diseases.

Accordingly, the proteins of the invention are useful for the diagnosisand/or treatment (including prevention) of immune related diseases.Antibodies which bind to stimulatory proteins are useful to suppress theinflammatory response. Antibodies which bind to inhibitory proteins areuseful to stimulate inflammatory response and the immune system. Theproteins and antibodies of the invention are also useful to preparemedicines and medicaments for the treatment of inflammatory and immunerelated diseases.

In one embodiment, the invention concerns antagonists and agonists of aPRO1868 polypeptide that inhibits one or more of the functions oractivities of a PRO1868 polypeptide.

In another embodiment, the invention concerns a method for determiningthe presence of a PRO1868 polypeptide comprising exposing a cellsuspected of containing the polypeptide to an anti-PRO1868 antibody anddetermining binding of the antibody to the cell.

In yet another embodiment, the present invention relates to a method ofdiagnosing an inflammatory elated disease in a mammal, comprisingdetecting the level of expression of a gene encoding a PRO1868polypeptide (a) in a test sample of tissue cells obtained from themammal, and (b) in a control sample of known normal tissue cells of thesame cell type, wherein a higher expression level in the test sampleindicates the presence of an inflammatory disease in the mammal.

In another embodiment, the present invention relates to method ofdiagnosing an inflammatory disease in a mammal, comprising (a)contacting an anti-PRO1868 antibody with a test sample of tissue culturecells obtained from the mammal, and (b) detecting the formation of acomplex between the antibody and the PRO1868 polypeptide. The detectionmay be qualitative or quantitative, and may be performed in comparisonwith monitoring the complex formation in a control sample of knownnormal tissue cells of the same cell type. A larger quantity ofcomplexes formed in the test sample indicates the presence of tumor inthe mammal from which the test tissue cells were obtained. The antibodypreferably carries a detectable label. Complex formation can bemonitored, for example, by light microscopy, flow cytometry,fluorimetry, or other techniques known in the art. The test sample isusually obtained from an individual suspected of having a deficiency orabnormality relating to the inflammatory response.

In another embodiment, the present invention relates to a diagnostickit, containing an anti-PRO1868 antibody and a carrier (e.g., a buffer)in suitable packaging. The kit preferably contains instructions forusing the antibody to detect the PRO1868 polypeptide.

In a further embodiment, the invention concerns an article ofmanufacture, comprising:

-   -   a container;    -   a label on the container; and    -   a composition comprising an active agent contained within the        container; wherein the composition is effective for stimulating        or inhibiting an inflammatory response in a mammal, the label on        the container indicates that the composition can be used to        treat an inflammatory disease, and the active agent in the        composition is an agent stimulating or inhibiting the expression        and/or activity of the PRO1868 polypeptide. In a preferred        aspect, the active agent is a PRO1868 polypeptide or an        anti-PRO1868 antibody.

A further embodiment is a method for identifying a compound capable ofinhibiting the expression and/or activity of a PRO1868 polypeptide bycontacting a candidate compound with a PRO1868 polypeptide underconditions and for time sufficient to allow these two compounds tointeract. In a specific aspect, either the candidate compound or thePRO1868 polypeptide is immobilized on a solid support. In anotheraspect, the non-immobilized component carries a detectable label.

In yet a further aspect, the invention relates to a method of treatingan inflammatory disease, by administration of an effective therapeuticamount of a PRO1868 antagonist to a patient in need thereof for thetreatment of a disease selected from: inflammatory bowel disease,systemic lupus erythematosis, rheumatoid arthritis, juvenile chronicarthritis, spondyloarthropathies, systemic sclerosis (scleroderma),idiopathic inflammatory myopathies (dermatomyositis, polymyositis),Sjogren's syndrome, systemic vaculitis, sarcoidosis, autoimmunehemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria), autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis),demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic polyneuropathy, hepatobiliarydiseases such as infectious hepatitis (hepatitis A, B, C, D, E and othernonhepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory and fibrotic lung diseases (e.g., cystic fibrosis,eosinophilic pneumonias, idiopathic pulmonary fibrosis andhypersensitivity pneumonitis), gluten-sensitive enteropathy, Whipple'sdisease, autoimmune or immune-mediated skin diseases including bullousskin diseases, erythema multiforme and contact dermatitis, psoriasis,allergic diseases of the lung such as eosinophilic pneumonias,idiopathic pulmonary fibrosis and hypersensitivity pneumonitis,transplantation associated diseases including graft rejection andgraft-verus host disease.

In a further embodiment, the present invention provides a method ofdiagnosing tumor in a mammal, comprising detecting the level ofexpression of a gene encoding a PRO1868 polypeptide (a) in a test sampleof tissue cells obtained from the mammal, and (b) in a control sample ofknown normal tissue cells of the same cell type, wherein a higherexpression level in the test sample indicates the presence of tumor inthe mammal from which the test tissue cells were obtained.

In another embodiment, the present invention provides a method ofdiagnosing tumor in a mammal, comprising (a) contacting an anti-PRO1868antibody with a test sample of the tissue cells obtained from themammal, and (b) detecting the formation of a complex between theanti-PRO1868 and the PRO1868 polypeptide in the test sample. Thedetection may be qualitative or quantitative, and may be performed incomparison with monitoring the complex formation in a control sample ofknown normal tissue cells of the same cell type. A larger quantity ofcomplexes formed in the test sample indicates the presence of tumor inthe mammal from which the test tissue cells were obtained. The antibodypreferably carries a detectable label. Complex formation can bemonitored, for example, by light microscopy, flow cytometry,fluorimetry, or other techniques known in the art. Preferably, the testsample is obtained from an individual mammal suspected to haveneoplastic cell growth or proliferation (e.g., cancerous cells).

In another embodiment, the present invention provides a cancerdiagnostic kit, comprising an anti-PRO1868 antibody and a carrier (e.g.a buffer) in suitable packaging. The kit preferably containsinstructions for using the antibody to detect the PRO1868 polypeptide.

In yet another embodiment, the invention provides a method forinhibiting the growth of tumor cells comprising exposing a cell whichoverexpresses a PRO1868 polypeptide to an effective amount of an agentinhibiting the expression and/or activity of the PRO1868 polypeptide.The agent preferably is an anti-PRO1868 polypeptide, a small organic andinorganic peptide, phosphopeptide, antisense or ribozyme molecule, or atriple helix molecule. In a specific aspect, the agent, e.g.,anti-PRO1868 antibody induces cell death. In a further aspect, the tumorcells are further exposed to radiation treatment and/or a cytotoxic orchemotherapeutic agent.

In a further embodiment, the invention concerns an article ofmanufacture, comprising:

-   a container;-   a label on the container, and-   a composition comprising an active agent contained within the    container; wherein the composition is effective for inhibiting the    growth of tumor cells, the label on the container indicates that the    composition can be used for treating conditions characterized by    overexpression of a PRO1868 polypeptide, and the active agent in the    composition is an agent inhibiting the expression and/or activity of    the PRO1868 polypeptide. In a preferred aspect, the active agent is    an anti-PRO1868 antibody.

A cDNA clone (DNA77624-2515) has been identified, having homology tonucleic acid encoding A33 antigen, that encodes a novel polypeptide,designated in the present application as “PRO1868”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1868 polypeptide.

In one aspect, the isolated nucleic acid comprises DNA having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to (a) a DNA moleculeencoding a PRO1868 polypeptide having the sequence of amino acidresidues from about 1 or about 31 to about 310, inclusive of FIG. 14(SEQ ID NO:20), or (b) the complement of the DNA molecule of (a).

In another aspect, the invention concerns an isolated nucleic acidmolecule encoding a PRO1868 polypeptide comprising DNA hybridizing tothe complement of the nucleic acid between about nucleotides 51 or about141 and about 980, inclusive, of FIG. 13 (SEQ ID NO:19). Preferably,hybridization occurs under stringent hybridization and wash conditions.

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA having at least about 80% sequence identity,preferably at least about 85% sequence identity, more preferably atleast about 90% sequence identity, most preferably at least about 95%sequence identity to (a) a DNA molecule encoding the same maturepolypeptide encoded by the human protein cDNA in ATCC Deposit No. 203553(DNA77624-2515) or (b) the complement of the nucleic acid molecule of(a). In a preferred embodiment, the nucleic acid comprises a DNAencoding the same mature polypeptide encoded by the human protein cDNAin ATCC Deposit No. 203553 (DNA77624-2515).

In still a further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to the sequence of aminoacid residues 1 or about 31 to about 310 inclusive of FIG. 14 (SEQ IDNO:20), or (b) the complement of the DNA of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule having at least 390 nucleotides and produced by hybridizing atest DNA molecule under stringent conditions with (a) a DNA moleculeencoding a PRO1868 polypeptide having the sequence of amino acidresidues from 1 or about 31 to about 310, inclusive of FIG. 14 (SEQ IDNO:20), or (b) the complement of the DNA molecule of (a), and, if theDNA molecule has at least about an 80% sequence identity, prefereably atleast about an 85% sequence identity, more preferably at least about a90% sequence identity, most preferably at least about a 95% sequenceidentity to (a) or (b), isolating the test DNA molecule.

In a specific aspect, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1868 polypeptide, with or withoutthe N-terminal signal sequence and/or the initiating methionine, and itssoluble, i.e., transmembrane domain deleted or inactivated variants, oris complementary to such encoding nucleic acid molecule. The signalpeptide has been tentatively identified as extending from about aminoacid position 1 to about amino acid position 30 in the sequence of FIG.14 (SEQ ID NO:20). The transmembrane domain has been tentativelyidentified as extending from about amino acid position 243 to aboutamino acid position 263 in the PRO1868 amino acid sequence (FIG. 14, SEQID NO:20).

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1or about 31 to about 310 inclusive of FIG. 14 (SEQ ID NO:20), or (b) thecomplement of the DNA of (a).

Another embodiment is directed to fragments of a PRO1868 polypeptidecoding sequence that may find use as hybridization probes. Such nucleicacid fragments are from about 20 to about 80 nucleotides in length,preferably from about 20 to about 60 nucleotides in length, morepreferably from about 20 to about 50 nucleotides in length and mostpreferably from about 20 to about 40 nucleotides in length and may bederived from the nucleotide sequence shown in FIG. 13 (SEQ ID NO:19).

In another embodiment, the invention provides isolated PRO1868polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

In a specific aspect, the invention provides isolated native sequencePRO1868 polypeptide, which in certain embodiments, includes an aminoacid sequence comprising residues 1 or about 31 to about 310 of FIG. 14(SEQ ID NO:20).

In another aspect, the invention concerns an isolated PRO1868polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 or about 31 to about 310, inclusive of FIG. 14 (SEQ ID NO:20).

In a further aspect, the invention concerns an isolated PRO1868polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 orabout 31 to about 310, inclusive of FIG. 14 (SEQ ID NO:20).

In yet another aspect, the invention concerns an isolated PRO1868polypeptide, comprising the sequence of amino acid residues 1 or about31 to about 310, inclusive of FIG. 14 (SEQ ID NO:20), or a fragmentthereof sufficient to provide a binding site for an anti-PRO1868antibody. Preferably, the PRO1868 fragment retains a qualitativebiological activity of a native PRO1868 polypeptide.

In a still further aspect, the invention provides a polypeptide producedby (i) hybridizing a test DNA molecule under stringent conditions with(a) a DNA molecule encoding a PRO1868 polypeptide having the sequence ofamino acid residues from about 1 or about 31 to about 310, inclusive ofFIG. 14 (SEQ ID NO:20), or (b) the complement of the DNA molecule of(a), and if the test DNA molecule has at least about an 80% sequenceidentity, preferably at least about an 85% sequence identity, morepreferably at least about a 90% sequence identity, most preferably atleast about a 95% sequence identity to (a) or (b), (ii) culturing a hostcell comprising the test DNA molecule under conditions suitable forexpression of the polypeptide, and (iii) recovering the polypeptide fromthe cell culture.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO1868 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-PRO1868 antibody.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists of a native PRO1868 polypeptide by contactingthe native PRO1868 polypeptide with a candidate molecule and monitoringa biological activity mediated by said polypeptide.

In a still further embodiment, the invention concerns a compositioncomprising a PRO1868 polypeptide, or an agonist or antagonist ashereinabove defined, in combination with a pharmaceutically acceptablecarrier.

In another embodiment, the invention provides a composition containing aPRO1868 polypeptide or an agonist or antagonist antibody in admixturewith a carrier or excipient. In one aspect, the composition contains atherapeutically affective amount of the peptide or antibody. In anotheraspect, when the composition contains an inflammation stimulatingmolecule, the composition is useful for: (a) increasing infiltration ofinflammatory cells into a tissue of a mammal in need thereof, (b)stimulating or enhancing an immune response in a mammal in need thereof,or (c) increasing the proliferation of T-lymphocytes in a mammal in needthereof in response to an antigen. In a further aspect, when thecomposition contains an inflammatory inhibiting molecule, thecomposition is useful for: (a) decreasing infiltration of inflammatorycells into a tissue of a mammal in need thereof, (b) inhibiting orreducing an inflammatory response in a mammal in need thereof, or (c)decreasing the proliferation of T-lymphocytes in a mammal in needthereof in response to an antigen. In another aspect, the compositioncontains a further active ingredient, which may, for example, be afurther antibody or a cytotoxic or chemotherapeutic agent. Preferably,the composition is sterile.

In a further embodiment, the invention concerns nucleic acid encoding ananti-PRO1868 antibody, and vectors and recombinant host cells comprisingsuch nucleic acid. In a still further embodiment, the invention concernsa method for producing such an antibody by culturing a host celltransformed with nucleic acid encoding the antibody under conditionssuch that the antibody is expressed, and recovering the antibody fromthe cell culture.

8. PRO3434

A cDNA clone (DNA77631-2537) has been identified that encodes a novelpolypeptide, designated in the present application as “PRO3434.”

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO3434 polypeptide.

In one aspect, the isolated nucleic acid comprises DNA having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to (a) a DNA moleculeencoding a PRO3434 polypeptide having the sequence of amino acidresidues from 1 or about 17 to about 1029, inclusive of FIG. 16 (SEQ IDNO:22), or (b) the complement of the DNA molecule of (a).

In another aspect, the invention concerns an isolated nucleic acidmolecule encoding a PRO3434 polypeptide comprising DNA hybridizing tothe complement of the nucleic acid between about residues 46 or about 94and about 3132, inclusive, of FIG. 15 (SEQ ID NO:21). Preferably,hybridization occurs under stringent hybridization and wash conditions.

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA having at least about 80% sequence identity,preferably at least about 85% sequence identity, more preferably atleast about 90% sequence identity, most preferably at least about 95%sequence identity to (a) a DNA molecule encoding the same maturepolypeptide encoded by the human protein cDNA in ATCC Deposit No. 203651(DNA77631-2537), or (b) the complement of the DNA molecule of (a). In apreferred embodiment, the nucleic acid comprises a DNA encoding the samemature polypeptide encoded by the human protein cDNA in ATCC Deposit No.203651 (DNA77631-2537).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to the sequence of aminoacid residues from 1 or about 17 to about 1029, inclusive of FIG. 16(SEQ ID NO:22), or the complement of the DNA of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule having at least about 460 nucleotides and produced byhybridizing a test DNA molecule under stringent conditions with (a) aDNA molecule encoding a PRO3434 polypeptide having the sequence of aminoacid residues from 1 or about 17 to about 1029, inclusive of FIG. 16(SEQ ID NO:22), or (b) the complement of the DNA molecule of (a), and,if the DNA molecule has at least about an 80% sequence identity,preferably at least about an 85% sequence identity, more preferably atleast about a 90% sequence identity, most preferably at least about a95% sequence identity to (a) or (b), isolating the test DNA molecule.

In a specific aspect, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO3434 polypeptide, with or withoutthe N-terminal signal sequence and/or the initiating methionine, or iscomplementary to such encoding nucleic acid molecule. The signal peptidehas been tentatively identified as extending from amino acid position 1through about amino acid position 16 in the sequence of FIG. 16 (SEQ IDNO:22).

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1or about 17 to about 1029, inclusive of FIG. 16 (SEQ ID NO:22), or (b)the complement of the DNA of (a).

Another embodiment is directed to fragments of a PRO3434 polypeptidecoding sequence that may find use as hybridization probes. Such nucleicacid fragments are from about 20 to about 80 nucleotides in length,preferably from about 20 to about 60 nucleotides in length, morepreferably from about 20 to about 50 nucleotides in length, and mostpreferably from about 20 to about 40 nucleotides in length.

In another embodiment, the invention provides isolated PRO3434polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove defined.

In a specific aspect, the invention provides isolated native sequencePRO3434 polypeptide, which in one embodiment, includes an amino acidsequence comprising residues 1 or about 17 to 1029 of FIG. 16 (SEQ IDNO:22).

In another aspect, the invention concerns an isolated PRO3434polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 or about 17 to about 1029, inclusive of FIG. 16 (SEQ ID NO:22).

In a further aspect, the invention concerns an isolated PRO3434polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 orabout 17 to 1029 of FIG. 16 (SEQ ID NO:22).

In yet another aspect, the invention concerns an isolated PRO3434polypeptide, comprising the sequence of amino acid residues 1 or about17 to about 1029, inclusive of FIG. 16 (SEQ ID NO:22), or a fragmentthereof sufficient to provide a binding site for an anti-PRO3434tibody.Preferably, the PRO982 fragment retains a qualitative biologicalactivity of a native PRO3434 polypeptide.

In a still further aspect, the invention provides a polypeptide producedby (i) hybridizing a test DNA molecule under stringent conditions with(a) a DNA molecule encoding a PRO3434 polypeptide having the sequence ofamino acid residues from 1 or about 17 to about 1029, inclusive of FIG.16 (SEQ ID NO:22), or (b) the complement of the DNA molecule of (a), andif the test DNA molecule has at least about an 80% sequence identity,preferably at least about an 85% sequence identity, more preferably atleast about a 90% sequence identity, most preferably at least about a95% sequence identity to (a) or (b), (ii) culturing a host cellcomprising the test DNA molecule under conditions suitable forexpression of the polypeptide, and (iii) recovering the polypeptide fromthe cell culture.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO3434 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-PRO3434 antibody.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists of a native PRO3434 polypeptide, by contactingthe native PRO3434 polypeptide with a candidate molecule and monitoringa biological activity mediated by said polypeptide.

In a still further embodiment, the invention concerns a compositioncomprising a PRO3434 polypeptide, or an agonist or antagonist ashereinabove defined, in combination with a pharmaceutically acceptablecarrier.

9. PRO1927

A cDNA clone (DNA82307-2531)has been identified that encodes a novelpolypeptide having homology to glycosyltransferases, and is designatedin the present application as “PRO1927”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1927 polypeptide.

In one aspect, the isolated nucleic acid comprises DNA having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to (a) a DNA moleculeencoding a PRO1927 polypeptide having the sequence of amino acidresidues from 1 or about 24 to about 548, inclusive of FIG. 18 (SEQ IDNO:24), or (b) the complement of the DNA molecule of (a).

In another aspect, the invention concerns an isolated nucleic acidmolecule encoding a PRO1927 polypeptide comprising DNA hybridizing tothe complement of the nucleic acid between about residues 120 and about1694, inclusive, of FIG. 17 (SEQ ID NO:23). Preferably, hybridizationoccurs under stringent hybridization and wash conditions.

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA having at least about 80% sequence identity,preferably at least about 85% sequence identity, more preferably atleast about 90% sequence identity, most preferably at least about 95%sequence identity to (a) a DNA molecule encoding the same maturepolypeptide encoded by the human protein cDNA in ATCC Deposit No. 203537(DNA82307-2531), or (b) the complement of the DNA molecule of (a). In apreferred embodiment, the nucleic acid comprises a DNA encoding the samemature polypeptide encoded by the human protein cDNA in ATCC Deposit No.203537 (DNA82307-2531).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to the sequence of aminoacid residues from 1 or about 24 to about 548, inclusive of FIG. 18 (SEQID NO:24), or the complement of the DNA of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule having at least about 50 nucleotides, and preferably at leastabout 100 nucleotides and produced by hybridizing a test DNA moleculeunder stringent conditions with (a) a DNA molecule encoding a PRO1927polypeptide having the sequence of amino acid residues from 1 or about24 to about 548, inclusive of FIG. 18 (SEQ ID NO:24), or (b) thecomplement of the DNA molecule of (a), and, if the DNA molecule has atleast about an 80% sequence identity, preferably at least about an 85%sequence identity, more preferably at least about a 90% sequenceidentity, most preferably at least about a 95% sequence identity to (a)or (b), isolating the test DNA molecule.

In a specific aspect, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1927 polypeptide, with or withoutthe N-terminal signal sequence and/or the initiating methionine, and itssoluble variants (i.e. transmembrane domain deleted or inactivated), oris complementary to such encoding nucleic acid molecule. The signalpeptide has been tentatively identified as extending from amino acidposition 1 through about amino acid position 23 in the sequence of FIG.18 (SEQ ID NO:24). A type II transmembrane domain has been tentativelyidentified as extending from about amino acid position 6 to about aminoacid position 25 in the PRO1927 amino acid sequence (FIG. 18, SEQ IDNO:24).

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1or about 24 to about 548, inclusive of FIG. 18 (SEQ ID NO:24), or (b)the complement of the DNA of (a).

Another embodiment is directed to fragments of a PRO1927 polypeptidecoding sequence that may find use as hybridization probes. Such nucleicacid fragments are from about 20 to about 80 nucleotides in length,preferably from about 20 to about 60 nucleotides in length, morepreferably from about 20 to about 50 nucleotides in length, and mostpreferably from about 20 to about 40 nucleotides in length.

In another embodiment, the invention provides isolated PRO1927polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove defined.

In a specific aspect, the invention provides isolated native sequencePRO1927 polypeptide, which in one embodiment, includes an amino acidsequence comprising residues 1 or about 24 to 548 of FIG. 18 (SEQ IDNO:24).

In another aspect, the invention concerns an isolated PRO1927polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 or about 24 to about 548, inclusive of FIG. 18 (SEQ ID NO:24).

In a further aspect, the invention concerns an isolated PRO1927polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 orabout 24 to 548 of FIG. 18 (SEQ ID NO:24).

In yet another aspect, the invention concerns an isolated PRO1927polypeptide, comprising the sequence of amino acid residues 1 or about24 to about 548, inclusive of FIG. 18 (SEQ ID NO:24), or a fragmentthereof sufficient to provide a binding site for an anti-PRO1927antibody. Preferably, the PRO1927 fragment retains a qualitativebiological activity of a native PRO1927 polypeptide.

In a still further aspect, the invention provides a polypeptide producedby (i) hybridizing a test DNA molecule under stringent conditions with(a) a DNA molecule encoding a PRO1927 polypeptide having the sequence ofamino acid residues from 1 or about 24 to about 548, inclusive of FIG.18 (SEQ ID NO:24), or (b) the complement of the DNA molecule of (a), andif the test DNA molecule has at least about an 80% sequence identity,preferably at least about an 85% sequence identity, more preferably atleast about a 90% sequence identity, most preferably at least about a95% sequence identity to (a) or (b), (ii) culturing a host cellcomprising the test DNA molecule under conditions suitable forexpression of the polypeptide, and (iii) recovering the polypeptide fromthe cell culture.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO1927 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-PRO1927 antibody.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists of a native PRO1927 polypeptide, by contactingthe native PRO1927 polypeptide with a candidate molecule and monitoringa biological activity mediated by said polypeptide.

In a still further embodiment, the invention concerns a compositioncomprising a PRO1927 polypeptide, or an agonist or antagonist ashereinabove defined, in combination with a pharmaceutically acceptablecarrier

10. Additional Embodiments

In other embodiments of the present invention, the invention providesvectors comprising DNA encoding any of the herein describedpolypeptides. Host cell comprising any such vector are also provided. Byway of example, the host cells may be CHO cells, E. coli, or yeast. Aprocess for producing any of the herein described polypeptides isfurther provided and comprises culturing host cells under conditionssuitable for expression of the desired polypeptide and recovering thedesired polypeptide from the cell culture.

In other embodiments, the invention provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence. Example of suchchimeric molecules comprise any of the herein described polypeptidesfused to an epitope tag sequence or a Fc region of an immunoglobulin.

In another embodiment, the invention provides an antibody whichspecifically binds to any of the above or below described polypeptides.Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody.

In yet other embodiments, the invention provides oligonucleotide probesuseful for isolating genomic and cDNA nucleotide sequences or asantisense probes, wherein those probes may be derived from any of theabove or below described nucleotide sequences.

In other embodiments, the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence that encodes a PROpolypeptide.

In one aspect, the isolated nucleic acid molecule comprises a nucleotidesequence having at least about 80% sequence identity, preferably atleast about 81% sequence identity, more preferably at least about 82%sequence identity, yet more preferably at least about 83% sequenceidentity, yet more preferably at least about 84% sequence identity, yetmore preferably at least about 85% sequence identity, yet morepreferably at least about 86% sequence identity, yet more preferably atleast about 87% sequence identity, yet more preferably at least about88% sequence identity, yet more preferably at least about 89% sequenceidentity, yet more preferably at least about 90% sequence identity, yetmore preferably at least about 91% sequence identity, yet morepreferably at least about 92% sequence identity, yet more preferably atleast about 93% sequence identity, yet more preferably at least about94% sequence identity, yet more preferably at least about 95% sequenceidentity, yet more preferably at least about 96% sequence identity, yetmore preferably at least about 97% sequence identity, yet morepreferably at least about 98% sequence identity and yet more preferablyat least about 99% sequence identity to (a) a DNA molecule encoding aPRO polypeptide having a full-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein, an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein, or (b) the complement of the DNA molecule of (a).

In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% sequence identity,preferably at least about 81% sequence identity, more preferably atleast about 82% sequence identity, yet more preferably at least about83% sequence identity, yet more preferably at least about 84% sequenceidentity, yet more preferably at least about 85% sequence identity, yetmore preferably at least about 86% sequence identity, yet morepreferably at least about 87% sequence identity, yet more preferably atleast about 88% sequence identity, yet more preferably at least about89% sequence identity, yet more preferably at least about 90% sequenceidentity, yet more preferably at least about 91% sequence identity, yetmore preferably at least about 92% sequence identity, yet morepreferably at least about 93% sequence identity, yet more preferably atleast about 94% sequence identity, yet more preferably at least about95% sequence identity, yet more preferably at least about 96% sequenceidentity, yet more preferably at least about 97% sequence identity, yetmore preferably at least about 98% sequence identity and yet morepreferably at least about 99% sequence identity to (a) a DNA moleculecomprising the coding sequence of a full-length PRO polypeptide cDNA asdisclosed herein, the coding sequence of a PRO polypeptide lacking thesignal peptide as disclosed herein, the coding sequence of anextracellular domain of a transmembrane PRO polypeptide, with or withoutthe signal peptide, as disclosed herein or the coding sequence of anyother specifically defined fragment of the full-length amino acidsequence as disclosed herein, or (b) the complement of the DNA moleculeof (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence having at least about 80%sequence identity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to (a) a DNAmolecule that encodes the same mature polypeptide encoded by any of thehuman protein cDNAs deposited with the ATCC as disclosed herein, or (b)the complement of the DNA molecule of (a).

Another aspect the invention provides an isolated nucleic acid moleculecomprising a nucleotide sequence encoding a PRO polypeptide which iseither transmembrane domain-deleted or transmembrane domain-inactivated,or is complementary to such encoding nucleotide sequence, wherein thetransmembrane domain(s) of such polypeptide are disclosed herein.Therefore, soluble extracellular domains of the herein described PROpolypeptides are contemplated.

Another embodiment is directed to fragments of a PRO polypeptide codingsequence, or the complement thereof, that may find use as, for example,hybridization probes, for encoding fragments of a PRO polypeptide thatmay optionally encode a polypeptide comprising a binding site for ananti-PRO antibody or as antisense oligonucleotide probes. Such nucleicacid fragments are usually at least about 20 nucleotides in length,preferably at least about 30 nucleotides in length, more preferably atleast about 40 nucleotides in length, yet more preferably at least about50 nucleotides in length, yet more preferably at least about 60nucleotides in length, yet more preferably at least about 70 nucleotidesin length, yet more preferably at least about 80 nucleotides in length,yet more preferably at least about 90 nucleotides in length, yet morepreferably at least about 100 nucleotides in length, yet more preferablyat least about 110 nucleotides in length, yet more preferably at leastabout 120 nucleotides in length, yet more preferably at least about 130nucleotides in length, yet more preferably at least about 140nucleotides in length, yet more preferably at least about 150nucleotides in length, yet more preferably at least about 160nucleotides in length, yet more preferably at least about 170nucleotides in length, yet more preferably at least about 180nucleotides in length, yet more preferably at least about 190nucleotides in length, yet more preferably at least about 200nucleotides in length, yet more preferably at least about 250nucleotides in length, yet more preferably at least about 300nucleotides in length, yet more preferably at least about 350nucleotides in length, yet more preferably at least about 400nucleotides in length, yet more preferably at least about 450nucleotides in length, yet more preferably at least about 500nucleotides in length, yet more preferably at least about 600nucleotides in length, yet more preferably at least about 700nucleotides in length, yet more preferably at least about 800nucleotides in length, yet more preferably at least about 900nucleotides in length and yet more preferably at least about 1000nucleotides in length, wherein in this context the term “about” meansthe referenced nucleotide sequence length plus or minus 10% of thatreferenced length. It is noted that novel fragments of a PROpolypeptide-encoding nucleotide sequence may be determined in a routinemanner by aligning the PRO polypeptide-encoding nucleotide sequence withother known nucleotide sequences using any of a number of well knownsequence alignment programs and determining which PROpolypeptide-encoding nucleotide sequence fragment(s) are novel. All ofsuch PRO polypeptide-encoding nucleotide sequences are contemplatedherein. Also contemplated are the PRO polypeptide fragments encoded bythese nucleotide molecule fragments, preferably those PRO polypeptidefragments that comprise a binding site for an anti-PRO antibody.

In another embodiment, the invention provides isolated PRO polypeptideencoded by any of the isolated nucleic acid sequences hereinaboveidentified.

In a certain aspect, the invention concerns an isolated PRO polypeptide,comprising an amino acid sequence having at least about 80% sequenceidentity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to a PROpolypeptide having a full-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein, an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein.

In a further aspect, the invention concerns an isolated PRO polypeptidecomprising an amino acid sequence having at least about 80% sequenceidentity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to an aminoacid sequence encoded by any of the human protein cDNAs deposited withthe ATCC as disclosed herein.

In a further aspect, the invention concerns an isolated PRO polypeptidecomprising an amino acid sequence scoring at least about 80% positives,preferably at least about 81% positives, more preferably at least about82% positives, yet more preferably at least about 83% positives, yetmore preferably at least about 84% positives, yet more preferably atleast about 85% positives, yet more preferably at least about 86%positives, yet more preferably at least about 87% positives, yet morepreferably at least about 88% positives, yet more preferably at leastabout 89% positives, yet more preferably at least about 90% positives,yet more preferably at least about 91% positives, yet more preferably atleast about 92% positives, yet more preferably at least about 93%positives, yet more preferably at least about 94% positives, yet morepreferably at least about 95% positives, yet more preferably at leastabout 96% positives, yet more preferably at least about 97% positives,yet more preferably at least about 98% positives and yet more preferablyat least about 99% positives when compared with the amino acid sequenceof a PRO polypeptide having a full-length amino acid sequence asdisclosed herein, an amino acid sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a transmembrane protein,with or without the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein.

In a specific aspect, the invention provides an isolated PRO polypeptidewithout the N-terminal signal sequence and/or the initiating methionineand is encoded by a nucleotide sequence that encodes such an amino acidsequence as hereinbefore described. Processes for producing the same arealso herein described, wherein those processes comprise culturing a hostcell comprising a vector which comprises the appropriate encodingnucleic acid molecule under conditions suitable for expression of thePRO polypeptide and recovering the PRO polypeptide from the cellculture.

Another aspect the invention provides an isolated PRO polypeptide whichis either transmembrane domain-deleted or transmembranedomain-inactivated. Processes for producing the same are also hereindescribed, wherein those processes comprise culturing a host cellcomprising a vector which comprises the appropriate encoding nucleicacid molecule under conditions suitable for expression of the PROpolypeptide and recovering the PRO polypeptide from the cell culture.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO polypeptide as defined herein. In aparticular embodiment, the agonist or antagonist is an anti-PRO antibodyor a small molecule.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists to a PRO polypeptide which comprise contactingthe PRO polypeptide with a candidate molecule and monitoring abiological activity mediated by said PRO polypeptide. Preferably, thePRO polypeptide is a native PRO polypeptide.

In a still further embodiment, the invention concerns a composition ofmatter comprising a PRO polypeptide, or an agonist or antagonist of aPRO polypeptide as herein described, or an anti-PRO antibody, incombination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

Another embodiment of the present invention is directed to the use of aPRO polypeptide, or an agonist or antagonist thereof as hereinbeforedescribed, or an anti-PRO antibody, for the preparation of a medicamentuseful in the treatment of a condition which is responsive to the PROpolypeptide, an agonist or antagonist thereof or an anti-PRO antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native sequencePRO1800 cDNA, wherein SEQ ID NO:1 is a clone designated herein as“DNA35672-2508”.

FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived from thecoding sequence of SEQ ID NO:1 shown in FIG. 1.

FIG. 3 shows a nucleotide sequence (SEQ ID NO:6) of a native sequencePRO539 cDNA, wherein SEQ ID NO:6 is a clone designated herein as“DNA47465-1561”.

FIG. 4 shows the amino acid sequence (SEQ ID NO:7) derived from thecoding sequence of SEQ ID NO:6 shown in FIG. 3.

FIG. 5 shows a nucleotide sequence (SEQ ID NO:8) of a native sequencePRO982 cDNA, wherein SEQ ID NO:8 is a clone designated herein as“DNA57700-1408”.

FIG. 6 shows the amino acid sequence (SEQ ID NO:9) derived from thecoding sequence of SEQ ID NO: 8 shown in FIG. 5.

FIG. 7 shows a nucleotide sequence (SEQ ID NO:10) of a native sequencePRO1434 cDNA, wherein SEQ ID NO:10 is a clone designated herein as“DNA68818-2536”.

FIG. 8 shows the amino acid sequence (SEQ ID NO:11) derived from thecoding sequence of SEQ ID NO:10 shown in FIG. 7.

FIG. 9 shows a nucleotide sequence (SEQ ID NO:15) of a native sequencePRO1863 cDNA, wherein SEQ ID NO:15 is a clone designated herein as“DNA59847-2510”.

FIG. 10 shows the amino acid sequence (SEQ ID NO:16) derived from thecoding sequence of SEQ ID NO:16 shown in FIG. 9.

FIG. 11 shows a nucleotide sequence (SEQ ID NO:17) of a native sequencePRO1917 cDNA, wherein SEQ ID NO:17 is a clone designated herein as“DNA76400-2528”.

FIG. 12 shows the amino acid sequence (SEQ ID NO:18) derived from thecoding sequence of SEQ ID NO:18 shown in FIG. 11.

FIG. 13 shows a nucleotide sequence (SEQ ID NO:19) of a native sequencePRO1868 cDNA, wherein SEQ ID NO:19 is a clone designated herein as“DNA77624-2515”.

FIG. 14 shows the amino acid sequence (SEQ ID NO:20) derived from thecoding sequence of SEQ ID NO:19 shown in FIG. 13.

FIG. 15 shows a nucleotide sequence (SEQ ID NO:21) of a native sequencePRO3434 cDNA, wherein SEQ ID NO:21 is a clone designated herein as“DNA77631-2537”.

FIG. 16 shows the amino acid sequence (SEQ ID NO:22) derived from thecoding sequence of SEQ ID NO:21 shown in FIG. 15.

FIG. 17 shows a nucleotide sequence (SEQ ID NO:23) of a native sequencePRO1927 cDNA, wherein SEQ ID NO:23 is a clone designated herein as“DNA82307-2531”.

FIG. 18 shows the amino acid sequence (SEQ ID NO:24) derived from thecoding sequence of SEQ ID NO:23 shown in FIG. 17.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

The terms “PRO polypeptide” and “PRO” as used herein and whenimmediately followed by a numerical designation refer to variouspolypeptides, wherein the complete designation (i.e., PRO/number) refersto specific polypeptide sequences as described herein. The terms“PRO/number polypeptide” and “PRO/number” wherein the term “number” isprovided as an actual numerical designation as used herein encompassnative sequence polypeptides and polypeptide variants (which are furtherdefined herein). The PRO polypeptides described herein may be isolatedfrom a variety of sources, such as from human tissue types or fromanother source, or prepared by recombinant or synthetic methods.

A “native sequence PRO polypeptide” comprises a polypeptide having thesame amino acid sequence as the corresponding PRO polypeptide derivedfrom nature. Such native sequence PRO polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native sequence PRO polypeptide” specifically encompassesnaturally-occurring truncated or secreted forms of the specific PROpolypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence PRO polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acids sequences shown in theaccompanying figures. Start and stop codons are shown in bold font andunderlined in the figures. However, while the PRO polypeptide disclosedin the accompanying figures are shown to begin with methionine residuesdesignated herein as amino acid position 1 in the figures, it isconceivable and possible that other methionine residues located eitherupstream or downstream from the amino acid position 1 in the figures maybe employed as the starting amino acid residue for the PRO polypeptides.

The PRO polypeptide “extracellular domain” or “ECD” refers to a form ofthe PRO polypeptide which is essentially free of the transmembrane andcytoplasmic domains. Ordinarily, a PRO polypeptide ECD will have lessthan 1% of such transmembrane and/or cytoplasmic domains and preferably,will have less than 0.5% of such domains. It will be understood that anytransmembrane domains identified for the PRO polypeptides of the presentinvention are identified pursuant to criteria routinely employed in theart for identifying that type of hydrophobic domain. The exactboundaries of a transmembrane domain may vary but most likely by no morethan about 5 amino acids at either end of the domain as initiallyidentified herein. Optionally, therefore, an extracellular domain of aPRO polypeptide may contain from about 5 or fewer amino acids on eitherside of the transmembrane domain/extracellular domain boundary asidentified in the Examples or specification and such polypeptides, withor without the associated signal peptide, and nucleic acid encodingthem, are comtemplated by the present invention.

The approximate location of the “signal peptides” of the various PROpolypeptides disclosed herein are shown in the present specificationand/or the accompanying figures. It is noted, however, that theC-terminal boundary of a signal peptide may vary, but most likely by nomore than about 5 amino acids on either side of the signal peptideC-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that type ofamino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10: 1-6(1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These polypeptides, wherethe signal peptide is cleaved within no more than about 5 amino acids oneither side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

“PRO polypeptide variant” means an active PRO polypeptide as definedabove or below having at least about 80% amino acid sequence identitywith a full-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any other fragment ofa full-length PRO polypeptide sequence as disclosed herein. Such PROpolypeptide variants include, for instance, PRO polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the full-length native amino acid sequence. Ordinarily, aPRO polypeptide variant will have at least about 80% amino acid sequenceidentity, preferably at least about 81% amino acid sequence identity,more preferably at least about 82% amino acid sequence identity, morepreferably at least about 83% amino acid sequence identity, morepreferably at least about 84% amino acid sequence identity, morepreferably at least about 85% amino acid sequence identity, morepreferably at least about 86% amino acid sequence identity, morepreferably at least about 87% amino acid sequence identity, morepreferably at least about 88% amino acid sequence identity, morepreferably at least about 89% amino acid sequence identity, morepreferably at least about 90% amino acid sequence identity, morepreferably at least about 91% amino acid sequence identity, morepreferably at least about 92% amino acid sequence identity, morepreferably at least about 93% amino acid sequence identity, morepreferably at least about 94% amino acid sequence identity, morepreferably at least about 95% amino acid sequence identity, morepreferably at least about 96% amino acid sequence identity, morepreferably at least about 97% amino acid sequence identity, morepreferably at least about 98% amino acid sequence identity and mostpreferably at least about 99% amino acid sequence identity with afull-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of a full-length PRO polypeptide sequenceas disclosed herein. Ordinarily, PRO variant polypeptides are at leastabout 10 amino acids in length, often at least about 20 amino acids inlength, more often at least about 30 amino acids in length, more oftenat least about 40 amino acids in length, more often at least about 50amino acids in length, more often at least about 60 amino acids inlength, more often at least about 70 amino acids in length, more oftenat least about 80 amino acids in length, more often at least about 90amino acids in length, more often at least about 100 amino acids inlength, more often at least about 150 amino acids in length, more oftenat least about 200 amino acids in length, more often at least about 300amino acids in length, or more.

“Percent (%) amino acid sequence identity” with respect to the PROpolypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the specific PRO polypeptide sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, % aminoacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations using this method, Tables 2 and 3 5 demonstrate how tocalculate the % amino acid sequence identity of the amino acid sequencedesignated “Comparison Protein” to the amino acid sequence designated“PRO”, wherein “PRO” represents the amino acid sequence of ahypothetical PRO polypeptide of interest, “Comparison Protein”represents the amino acid sequence of a polypeptide against which the“PRO” polypeptide of interest is being compared, and “X, “Y” and “Z”each represent different hypothetical amino acid residues.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, % aminoacid sequence identity values may also be obtained as described below byusing the WU-BLAST-2 computer program (Altschul et al., Methods inEnzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parametersare set to the default values. Those not set to default values, i.e.,the adjustable parameters, are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11, and scoringmatrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequenceidentity value is determined by dividing (a) the number of matchingidentical amino acid residues between the amino acid sequence of the PROpolypeptide of interest having a sequence derived from the native PROpolypeptide and the comparison amino acid sequence of interest (i.e.,the sequence against which the PRO polypeptide of interest is beingcompared which may be a PRO variant polypeptide) as determined byWU-BLAST-2 by (b) the total number of amino acid residues of the PROpolypeptide of interest. For example, in the statement “a polypeptidecomprising an the amino acid sequence A which has or having at least 80%amino acid sequence identity to the amino acid sequence B”, the aminoacid sequence A is the comparison amino acid sequence of interest andthe amino acid sequence B is the amino acid sequence of the PROpolypeptide of interest.

Percent amino acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison programmay be downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 usesseveral search parameters, wherein all of those search parameters areset to default values including, for example, unmask=yes, strand=all,expected occurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

“PRO variant polynucleotide” or “PRO variant nucleic acid sequence”means a nucleic acid molecule which encodes an active PRO polypeptide asdefined below and which has at least about 80% nucleic acid sequenceidentity with a nucleotide acid sequence encoding a full-length nativesequence PRO polypeptide sequence as disclosed herein, a full-lengthnative sequence PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any other fragment ofa full-length PRO polypeptide sequence as disclosed herein. Ordinarily,a PRO variant polynucleotide will have at least about 80% nucleic acidsequence identity, more preferably at least about 81% nucleic acidsequence identity, more preferably at least about 82% nucleic acidsequence identity, more preferably at least about 83% nucleic acidsequence identity, more preferably at least about 84% nucleic acidsequence identity, more preferably at least about 85% nucleic acidsequence identity, more preferably at least about 86% nucleic acidsequence identity, more preferably at least about 87% nucleic acidsequence identity, more preferably at least about 88% nucleic acidsequence identity, more preferably at least about 89% nucleic acidsequence identity, more preferably at least about 90% nucleic acidsequence identity, more preferably at least about 91% nucleic acidsequence identity, more preferably at least about 92% nucleic acidsequence identity, more preferably at least about 93% nucleic acidsequence identity, more preferably at least about 94% nucleic acidsequence identity, more preferably at least about 95% nucleic acidsequence identity, more preferably at least about 96% nucleic acidsequence identity, more preferably at least about 97% nucleic acidsequence identity, more preferably at least about 98% nucleic acidsequence identity and yet more preferably at least about 99% nucleicacid sequence identity with a nucleic acid sequence encoding afull-length native sequence PRO polypeptide sequence as disclosedherein, a full-length native sequence PRO polypeptide sequence lackingthe signal peptide as disclosed herein, an extracellular domain of a PROpolypeptide, with or without the signal sequence, as disclosed herein orany other fragment of a full-length PRO polypeptide sequence asdisclosed herein. Variants do not encompass the native nucleotidesequence.

Ordinarily, PRO variant polynucleotides are at least about 30nucleotides in length, often at least about 60 nucleotides in length,more often at least about 90 nucleotides in length, more often at leastabout 120 nucleotides in length, more often at least about 150nucleotides in length, more often at least about 180 nucleotides inlength, more often at least about 210 nucleotides in length, more oftenat least about 240 nucleotides in length, more often at least about 270nucleotides in length, more often at least about 300 nucleotides inlength, more often at least about 450 nucleotides in length, more oftenat least about 600 nucleotides in length, more often at least about 900nucleotides in length, or more.

“Percent (%) nucleic acid sequence identity” with respect toPRO-encoding nucleic acid sequences identified herein is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the PRO nucleic acid sequence of interest, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. For purposes herein, however, % nucleicacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”,wherein “PRO-DNA” represents a hypothetical PRO-encoding nucleic acidsequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “PRO-DNA” nucleicacid molecule of interest is being compared, and “N”, “L” and “V” eachrepresent different hypothetical nucleotides.

Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, %nucleic acid sequence identity values may also be obtained as describedbelow by using the WU-BLAST-2 computer program (Altschul et al., Methodsin Enzymology 266:460480 (1996)). Most of the WU-BLAST-2 searchparameters are set to the default values. Those not set to defaultvalues, i.e., the adjustable parameters, are set with the followingvalues: overlap span=1, overlap fraction=0.125, word threshold (T)=11,and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleicacid sequence identity value is determined by dividing (a) the number ofmatching identical nucleotides between the nucleic acid sequence of thePRO polypeptide-encoding nucleic acid molecule of interest having asequence derived from the native sequence PRO polypeptide-encodingnucleic acid and the comparison nucleic acid molecule of interest (i.e.,the sequence against which the PRO polypeptide-encoding nucleic acidmolecule of interest is being compared which may be a variant PROpolynucleotide) as determined by WU-BLAST-2 by (b) the total number ofnucleotides of the PRO polypeptide-encoding nucleic acid molecule ofinterest. For example, in the statement “an isolated nucleic acidmolecule comprising a nucleic acid sequence A which has or having atleast 80% nucleic acid sequence identity to the nucleic acid sequenceB”, the nucleic acid sequence A is the comparison nucleic acid moleculeof interest and the nucleic acid sequence B is the nucleic acid sequenceof the PRO polypeptide-encoding nucleic acid molecule of interest.

Percent nucleic acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison programmay be downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 usesseveral search parameters, wherein all of those search parameters areset to default values including, for example, unmask=yes, strand=all,expected occurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for sequence comparisons,the % nucleic acid sequence identity of a given nucleic acid sequence Cto, with, or against a given nucleic acid sequence D (which canalternatively be phrased as a given nucleic acid sequence C that has orcomprises a certain % nucleic acid sequence identity to, with, oragainst a given nucleic acid sequence D) is calculated as follows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program NCBI-BLAST2 in that program's alignment of Cand D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C.

In other embodiments, PRO variant polynucleotides are nucleic acidmolecules that encode an active PRO polypeptide and which are capable ofhybridizing, preferably under stringent hybridization and washconditions, to nucleotide sequences encoding a full-length PROpolypeptide as disclosed herein. PRO variant polypeptides may be thosethat are encoded by a PRO variant polynucleotide.

The term “positives”, in the context of sequence comparison performed asdescribed above, includes residues in the sequences compared that arenot identical but have similar properties (e.g. as a result ofconservative substitutions, see Table 6 below). For purposes herein, the% value of positives is determined by dividing (a) the number of aminoacid residues scoring a positive value between the PRO polypeptide aminoacid sequence of interest having a sequence derived from the native PROpolypeptide sequence and the comparison amino acid sequence of interest(i.e., the amino acid sequence against which the PRO polypeptidesequence is being compared) as determined in the BLOSUM62 matrix ofWU-BLAST-2 by (b) the total number of amino acid residues of the PROpolypeptide of interest.

Unless specifically stated otherwise, the % value of positives iscalculated as described in the immediately preceding paragraph. However,in the context of the amino acid sequence identity comparisons performedas described for ALIGN-2 and NCBI-BLAST-2 above, includes amino acidresidues in the sequences compared that are not only identical, but alsothose that have similar properties. Amino acid residues that score apositive value to an amino acid residue of interest are those that areeither identical to the amino acid residue of interest or are apreferred substitution (as defined in Table 6 below) of the amino acidresidue of interest.

For amino acid sequence comparisons using ALIGN-2 or NCBI-BLAST2, the %value of positives of a given amino acid sequence A to, with, or againsta given amino acid sequence B (which can alternatively be phrased as agiven amino acid sequence A that has or comprises a certain % positivesto, with, or against a given amino acid sequence B) is calculated asfollows:100 times the fraction X/Ywhere X is the number of amino acid residues scoring a positive value asdefined above by the sequence alignment program ALIGN-2 or NCBI-BLAST2in that program's alignment of A and B, and where Y is the total numberof amino acid residues in B. It will be appreciated that where thelength of amino acid sequence A is not equal to the length of amino acidsequence B, the % positives of A to B will not equal the % positives ofB to A.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” PRO polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single anti-PRO monoclonal antibodies (includingagonist, antagonist, and neutralizing antibodies), anti-PRO antibodycompositions with polyepitopic specificity, single chain anti-PROantibodies, and fragments of anti-PRO antibodies (see below). The term“monoclonal antibody” as used herein refers to an antibody obtained froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5× SSC (0.75 M NaCl, 0.075 M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2× SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1× SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and %SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5× SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,followed by washing the filters in 1× SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a PRO polypeptide fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

“Active” or “activity” for the purposes herein refers to form(s) of aPRO polypeptide which retain a biological and/or an immunologicalactivity of native or naturally-occurring PRO, wherein “biological”activity refers to a biological function (either inhibitory orstimulatory) caused by a native or naturally-occurring PRO other thanthe ability to induce the production of an antibody against an antigenicepitope possessed by a native or naturally-occurring PRO and an“immunological” activity refers to the ability to induce the productionof an antibody against an antigenic epitope possessed by a native ornaturally-occurring PRO.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native PRO polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a native PROpolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, fragments or amino acid sequence variants of native PROpolypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. Methods for identifying agonists or antagonists of a PROpolypeptide may comprise contacting a PRO polypeptide with a candidateagonist or antagonist molecule and measuring a detectable change in oneor more biological activities normally associated with the PROpolypeptide.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.μIntermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc. Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label may be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

By “solid phase” is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.,controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a PRO polypeptide or antibody thereto) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

TABLE 1 /*  *  * C-C increased from 12 to 15  * Z is average of EQ  * Bis average of ND  * match with stop is _M; stop-stop = 0; J (joker)match = 0  */ #define _M −8 /* value of a match with a stop */ int_day[26][26] = { /*  A B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/ /* A */ { 2, 0, −2, 0, 0, −4, 1, −1, −1, 0, −1, −2, −1, 0, _M, 1, 0,−2, 1, 1, 0, 0, −6, 0, −3, 0}, /* B */ { 0, 3, −4, 3, 2, −5, 0, 1, −2,0, 0, −3, −2, 2, _M, −1, 1, 0, 0, 0, 0, −2, −5, 0, −3, 1}, /* C */ {−2,−4, 15, −5, −5, −4, −3, −3, −2, 0, −5, −6, −5, −4, _M, −3, −5, −4, 0,−2, 0, −2, −8, 0, 0, −5}, /* D */ {0, 3, −5, 4, 3, −6, 1, 1, −2, 0, 0,−4, −3, 2, _M, −1, 2, −1, 0, 0, 0, −2, −7, 0, −4, 2}, /* E */ {0, 2, −5,3, 4, −5, 0, 1, −2, 0, 0, −3, −2, 1, _M, −1, 2, −1, 0, 0, 0, −2, −7, 0,−4, 3}, /* F */ {−4, −5, −4, −6, −5, 9, −5, −2, 1, 0, −5, 2, 0, −4, _M,−5, −5, −4, −3, −3, 0, −1, 0, 0, 7, −5}, /* G */ { 1, 0, −3, 1, 0, −5,5, −2, −3, 0, −2, −4, −3, 0, _M, −1, −1, −3, 1, 0, 0, −1, −7, 0, −5, 0},/* H */ {−1, 1, −3, 1, 1, −2, −2, 6, −2, 0, 0, −2, −2, 2, _M, 0, 3, 2,−1, −1, 0, −2, −3, 0, 0, 2}, /* I */ {−1, −2, −2, −2, −2, 1, −3, −2, 5,0, −2, 2, 2, −2, _M, −2, −2, −2, −1, 0, 0, 4, −5, 0, −1, −2}, /* J */{0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0,0, 0, 0}, /* K */ {−1, 0, −5, 0, 0, −5, −2, 0, −2, 0, 5, −3, 0, 1, _M,−1, 1, 3, 0, 0, 0, −2, −3, 0, −4, 0}, /* L */ {−2, −3, −6, −4, −3, 2,−4, −2, 2, 0, −3, 6, 4, −3, _M, −3, −2, −3, −3 , −1, 0, 2, −2, 0, −1,−2} /* M */ {−1, −2, −5, −3, −2, 0, −3, −2, 2, 0, 0, 4, 6, −2, _M, −2,−1, 0, −2, −1, 0, 2, −4, 0, −2, −1}, /* N */ {0, 2, −4, 2, 1, −4, 0, 2,−2, 0, 1, −3, −2, 2, _M, −1, 1, 0, 1, 0, 0, −2, −4, 0, −2, 1}, /* O */{_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,}, /* P */ {1, −1, −3, −1, −1, −5,−1, 0, −2, 0, −1, −3, −2, −1,_M, 6, 0, 0, 1, 0, 0, −1, −6, 0, −5, 0}, /*Q */ {0, 1, −5, 2, 2, −5, −1, 3, −2, 0, 1, −2, −1, 1, _M, 0, 4, 1, −1,−1, 0, −2, −5, 0, −4, 3}, /* R */ {−2, 0, −4, −1, −1, −4, −3, 2, −2, 0,3, −3, 0, 0, _M, 0, 1, 6, 0, −1, 0, −2, 2, 0, −4, 0}, /* S */ { 1, 0, 0,0, 0, −3, 1, −1, −1, 0, 0, −3, −2, 1, _M, 1, −1, 0, 2, 1, 0, −1, −2, 0,−3, 0}, /* T */ { 1, 0, −2, 0, 0, −3, 0, −1, 0, 0, 0, −1, −1, 0, _M, 0,−1, −1, 1, 3, 0, 0, −5, 0, −3, 0}, /* U */ {0, 0, 0, 0, 0, 0, 0, 0, 0,0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* V */ {0, −2, −2,−2, −2, −1, −1, −2, 4, 0, −2, 2, 2, −2,_M, −1, −2, −2, −1, 0, 0, 4, −6,0, −2, −2}, /* W */ {−6, −5, −8, −7, −7, 0, −7, −3, −5, 0, −3, −2, −4,−4,_M, −6, −5, 2, −2, −5, 0, −6, 17, 0, 0, −6}, /* X */ {0, 0, 0, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* Y */{−3, −3, 0, −4, −4, 7, −5, 0, −1, 0, −4, −1, −2, −2, _M, −5, −4, −4, −3,−3, 0, −2, 0, 0, 10, −4}, /* Z */ { 0, 1, −5, 2, 3, −5, 0, 2, −2, 0, 0,−2, −1, 1,_M, 0, 3, 0, 0, 0, 0, −2, −6, 0, −4, 4} }; /*  */ #include<stdio.h> #include <ctype.h> #define MAXJMP  16 /* max jumps in a diag*/ #define MAXGAP  24 /* don't continue to penalize gaps larger thanthis */ #define JMPS 1024 /* max jmps in an path */ #define MX   4 /*save if there's at least MX-1 bases since last jmp */ #define DMAT   3/* value of matching bases */ #define DMIS   0 /* penalty for mismatchedbases */ #define DINS0   8 /* penalty for a gap */ #define DINS1   1 /*penalty per base */ #define PINS0   8 /* penalty for a gap */ #definePINS1   4 /* penalty per residue */ struct jmp { short n[MAXJMP]; /*size of jmp (neg for dely) */ unsigned short x[MAXJMP]; /* base no. ofjmp in seq x */ /* limits seq to 2{circumflex over ( )}16 −1 */ };struct diag { int score; /* score at last jmp */ long offset; /* offsetof prev block */ short ijmp; /* current jmp index */ struct jmp jp; /*list of jmps */ }; struct path { int spc; /* number of leading spaces */short n[JMPS];/* size of jmp (gap) */ int x[JMPS];/* loc of jmp (lastelem before gap) */ }; char *ofile; /* output file name */ char*namex[2]; /* seq names: getseqs() */ char *prog; /* prog name for errmsgs */ char *seqx[2]; /* seqs: getseqs() */ int dmax; /* best diag:nw() */ int dmax0; /* final diag */ int dna; /* set if dna: main() */int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* totalgaps in seqs */ int len0, len1; /* seq lens */ int ngapx, ngapy; /*total size of gaps */ int smax; /* max score: nw() */ int *xbm; /*bitmap for matching */ long offset; /* current offset in jmp file */struct diag *dx; /* holds diagonals */ struct path pp[2]; /* holds pathfor seqs */ char *calloc(), *malloc(), *index(), *strcpy(); char*getseq(), *g_calloc(); /* Needleman-Wunsch alignment program  *  *usage: progs file1 file2  * where file1 and file2 are two dna or twoprotein sequences.  * The sequences can be in upper- or lower-case anmay contain ambiguity  * Any lines beginning with ‘;’, ‘>’ or ‘<’ areignored  * Max file length is 65535 (limited by unsigned short x in thejmp struct)  * A sequence with ⅓ or more of its elements ACGTU isassumed to be DNA  * Output is in the file “align.out”  *  * The programmay create a tmp file in /tmp to hold info about traceback.  * Originalversion developed under BSD 4.3 on a vax 8650  */ #include “nw.h”#include “day.h” static _dbval[26] = {1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static_pbval[26] = { 1, 2|(1< <(‘D’-‘A’))|(1< <(‘N’-‘A’)), 4, 8, 16, 32, 64,128, 256, 0xFFFFFFF, 1< <10, 1< <11, 1< <12, 1< <13, 1< <14, 1< <15, 1<<16, 1< <17, 1< <18, 1< <19, 1< <20, 1< <21, 1< <22, 1< <23, 1< <24, 1<<25|(1< <(‘E’-‘A’))|(1< <(‘Q’-‘A’)) }; main(ac, av) main int ac; char*av[]; { prog = av[0]; if(ac != 3) { fprintf(stderr, “usage: %s file1file2\n”, prog); fprintf(stderr, “where file1 and file2 are two dna ortwo protein sequences.\n”); fprintf(stderr, “The sequences can be inupper- or lower-case\n”); fprintf(stderr, “Any lines beginning with ‘;’or ‘<’ are ignored\n”); fprintf(stderr, “Output is in the file\“align.out\”\n”); exit(1); } namex[0] = av[1]; namex[1] = av[2];seqx[0] = getseq(namex[0], &len0); seqx[1] = getseq(namex[1], &len1);xbm = (dna)? _dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ofile = “align.out”; /* output file */ nw(); /* fill in the matrix, getthe possible jmps */ readjmps(); /* get the actual jmps */ print(); /*print stats, alignment */ cleanup(0); /* unlink any tmp files */ } /* dothe alignment, return best score: main()  * dna: values in Fitch andSmith, PNAS, 80, 1382-1386, 1983  * pro: PAM 250 values  * When scoresare equal, we prefer mismatches to any gap, prefer  * a new gap toextending an ongoing gap, and prefer a gap in seqx  * to a gap in seq y. */ nw() nw { char *px, *py; /* seqs and ptrs */ int *ndely, *dely; /*keep track of dely */ int ndelx, delx; /* keep track of delx */ int*tmp; /* for swapping row0, row1 */ int mis; /* score for each type */int ins0, ins1; /* insertion penalties */ register id; /* diagonal index*/ register ij; /* jmp index */ register *col0, *col1; /* score forcurr, last row */ register xx, yy; /* index into seqs */ dx = ( structdiag *)g_calloc(“to get diags”, len0 + len1 + 1, sizeof(struct diag));ndely = (int *)g_calloc(“to get ndely”, len1 + 1, sizeof(int)); dely =(int *)g_calloc(“to get dely”, len1 + 1, sizeof(int)); col0 = (int*)g_calloc(“to get col0”, len1 + 1, sizeof(int)); col1 = (int*)g_calloc(“to get col1”, len1 + 1, sizeof(int)); ins0 = (dna)? DINS0 :PINS0; ins1 = (dna)? DINS1 : PlNS1; smax = −10000; if (endgaps) { for(col0[0] = dely[0] = −ins0, yy = 1; yy <= len1; yy++) { col0[yy] =dely[yy] = col0[yy−1] − ins1; ndely[yy] = yy; } col0[0] = 0; /* WatermanBull Math Biol 84 */ } else for (yy = 1; yy <= len1; yy++) dely[yy] =−ins0; /* fill in match matrix  */ for (px = seqx[0], xx = 1; xx <=len0; px++, xx++) { /* initialize first entry in col  */ if (endgaps) {if (xx == 1) col1[0] = delx = −(ins0 + ins1); else col1[0] = delx =col0[0] − ins1; ndelx = xx; } else { col1[0] = 0; delx = −ins0; ndelx =0; } ...nw for (py = seqx[1], yy = 1; yy <= len1; py++, yy++) { mis =col0[yy−1]; if (dna) mis + = (xbm[*px−‘A’]&xbm[*py−‘A’])? DMAT : DMIS;else mis += _day[*px−‘A’][*py−‘A’]; /* update penalty for del in x seq; * favor new del over ongong del  * ignore MAXGAP if weighting endgaps */ if (endgaps || ndely[yy] < MAXGAP) { if (col0[yy] − ins0 >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } else {dely[yy] −= ins1; ndely[yy]++; } } else { if (col0[yy] − (ins0+ins1) >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } elsendely[yy]++; } /* update penalty for del in y seq;  * favor new del overongong del  */ if (endgaps || ndelx < MAXGAP) { if (col1[yy−1] − ins0 >=delx) { delx = col1[yy−1] − (ins0+ins1); ndelx = 1; } else { delx −=ins1; ndelx++; } } else { if (col1[yy−1] − (ins0+ins1) > = delx) { delx= col1[yy−1] − (ins0+ins1); ndelx = 1; } else ndelx+ +; } /* pick themaximum score; we're favoring  * mis over any del and delx over dely  */...nw id = xx − yy + len1 − 1; if (mis >= delx && mis >= dely[yy])col1[yy] = mis; else if (delx > = dely[yy]) { col1[yy] = delx; ij =dx[id].ijmp; if (dx[id].jp.n[0] && (!dna || (ndelx > = MAXJMP && xx >dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) { dx[id].ijmp+ +; if(++ij >= MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset =offset; offset += sizeof(struct jmp) + sizeof(offset); } }dx[id].jp.n[ij] = ndelx; dx[id].jp.x[ij] = xx; dx[id].score = delx; }else { col1[yy] = dely[yy]; ij = dx[id].ijmp; if (dx[id].jp.n[0] &&(!dna || (ndely[yy] > = MAXJMP && xx > dx[id].jp.x[ij]+MX) || mis >dx[id].score+DINS0)) { dx[id].ijmp ++; if (++ij >= MAXJMP) {writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset +=sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] = −ndely[yy];dx[id].jp.x[ij] = xx; dx[id].score = dely[yy]; } if (xx == len0 && yy <len1) { /* last col  */ if (endgaps) col1[yy] −= ins0+ins1*(len1−yy);if(col1[yy] > smax) { smax = col1[yy]; dmax = id; } } } if (endgaps &&xx < len0) col1[yy−1] −= ins0+ins1*(len0−xx); if (col1[yy−1] > smax) {smax = col1[yy−1]; dmax = id; } tmp = col0; col0 = col1; col1 = tmp;  } (void) free((char *)ndely);  (void) free((char *)dely);  (void)free((char *)col0);  (void) free((char *)col1); } /*  *  * print() --only routine visible outside this module  *  * static:  * getmat() --trace back best path, count matches: print()  * pr_align() -- printalignment of described in array p[]: print()  * dumpblock() -- dump ablock of lines with numbers, stars: pr_align()  * nums() -- put out anumber line: dumpblock()  * putline() -- put out a line (name, [num],seq, [num]): dumpblock()  * stars() - -put a line of stars: dumpblock() * stripname() -- strip any path and prefix from a seqname  */ #include“nw.h” #define SPC  3 #define P_LINE 256 /* maximum output line */#define P_SPC  3 /* space between name or num and seq */ extern_day[26][26]; int olen; /* set output line length */ FILE *fx; /* outputfile */ print() print { int lx, ly, firstgap, lastgap;  /* overlap */ if((fx = fopen(ofile, “w”)) == 0) { fprintf(stderr, “ %s: can't write%s\n”, prog, ofile); cleanup(1); } fprintf(fx, “<first sequence: %s(length = %d)\n”, namex[0], len0); fprintf(fx, “<second sequence: %s(length = %d)\n”, namex[1], len1); olen = 60; lx = len0; ly = len1;firstgap = lastgap = 0; if (dmax < len1 − 1) { /* leading gap in x */pp[0].spc = firstgap = len1 − dmax − 1; ly −= pp[0].spc; } else if(dmax > len1 − 1) { /* leading gap in y */ pp[1].spc = firstgap = dmax −(len1 − 1); lx −= pp[1].spc; } if (dmax0 < len0 − 1) { /* trailing gapin x */ lastgap = len0 − dmax0 −1; lx −= lastgap; } else if (dmax0 >len0 − 1) { /* trailing gap in y */ lastgap = dmax0 − (len0 − 1); ly −=lastgap; } getmat(lx, ly, firstgap, lastgap); pr_align(); } /*  * traceback the best path, count matches  */ static getmat(lx, ly, firstgap,lastgap) getmat int lx, ly; /* “core” (minus endgaps) */ int firstgap,lastgap; /* leading trailing overlap */ { int nm, i0, i1, siz0, siz1;char outx[32]; double pct; register n0, n1; register char *p0, *p1; /*get total matches, score  */ i0 = i1 = siz0 = siz1 = 0; p0 = seqx[0] +pp[1].spc; p1 = seqx[1] + pp[0].spc; n0 = pp[1].spc + 1; n1 =pp[0].spc + 1; nm = 0; while ( *p0 && *p1 ) { if (siz0) { p1++; n1++;siz0−−; } else if (siz1) { p0+ +; n0+ +; siz1−− } else { if(xbm[*p0−‘A’]&xbm[*p1−‘A’]) nm+ +; if (n0++ == pp[0].x[i0]) siz0 =pp[0].n[i0++]; if (nl++ == pp[1].x[i1]) siz1 = pp[1].n[il++]; p0+ +;p1++; } } /* pct homology:  * if penalizing endgaps, base is the shorterseq  * else, knock off overhangs and take shorter core  */ if (endgaps)lx = (len0 < len1)? len0 : len1; else lx = (lx < ly)? lx : ly; pct =100.*(double)nm/(double)lx; fprintf(fx, “\n”); fprintf(fx, “< %d match%sin an overlap of %d: %.2f percent similarity\n”, nm, (nm == 1)? “” :“es”, lx, pct); fprintf(fx, “<gaps in first sequence: %d”, gapx);...getmat if (gapx) { (void) sprintf(outx, “ (%d %s%s)”, ngapx, (dna)?“base”: “residue”, (ngapx == 1)? “”:“s”); fprintf(fx, “% s”, outx);fprintf(fx, “, gaps in second sequence: %d”, gapy); if (gapy) { (void)sprintf(outx, “(%d %s%s)”, ngapy, (dna)? “base”:“residue”, (ngapy == 1)?“”:“s”); fprintf(fx, “%s”, outx); } if (dna) fprintf(fx, “\n<score: %d(match = %d, mismatch = %d, gap penalty = %d + %d per base)\n”, smax,DMAT, DMIS, DINS0, DINS1); else fprintf(fx, “\n< score: %d (Dayhoff PAM250 matrix, gap penalty = %d + %d per residue)\n”, smax, PINS0, PINS1);if (endgaps) fprintf(fx, “<endgaps penalized. left endgap: %d %s%s,right endgap: %d %s%s\n”, firstgap, (dna)? “base” : “residue”, (firstgap== 1)? “” : “s”, lastgap, (dna)? “base” : “residue”, (lastgap == 1)? “”: “s”); else fprintf(fx, “<endgaps not penalized\n”); } static nm; /*matches in core -- for checking */ static lmax; /* lengths of strippedfile names */ static ij[2]; /* jmp index for a path */ static nc[2]; /*number at start of current line */ static ni[2]; /* current elem number-- for gapping */ static siz[2]; static char *ps[2]; /* ptr to currentelement */ static char *po[2]; /* ptr to next output char slot */ staticchar out[2][P_LINE]; /* output line */ static char star[P_LINE]; /* setby stars() */ /*  * print alignment of described in struct path pp[]  */static pr_align() pr_align { int nn; /* char count */ int more; registeri; for (i = 0, lmax = 0; i < 2++) { nn = stripname(namex[i]); if (nn >lmax) lmax = nn; nc[i] = 1; ni[i] = 1; siz[i] = ij[i] = 0; ps[i] =seqx[i]; po[i] = out[i]; } for (nn = nm = 0, more = 1; more;) {...pr_align for (i = more = 0; i < 2; i++) { /*  * do we have more ofthis sequence?  */ if (!*ps[i]) continue; more ++; if (pp[i].spc) { /*leading space */ *po[i]++ = ‘ ’; pp[i] .spc−−; } else if (siz[i]) { /*in a gap */ *po[i]++ = ‘−’; siz[i]−−; } else { /* we're putting a seqelement */ *po[i] = *ps[i]; if (islower(*ps[i])) *ps[i] =toupper(*ps[i]); po[i]++; ps[i]++; /*  * are we at next gap for thisseq?  */ if (ni[i] == pp[i].x[ij[i]]) { /*  * we need to merge all gaps * at this location  */ siz[i] == pp[i].n[ij[i]++]; while (ni[i] ==pp[i].x[ij[i]]) siz[i] += pp[i].n[ij[i] + +]; } ni[i] + +; } } if (++nn== olen || !more && nn) { dumpblock(); for (i = 0; i < 2; i++) po[i] =out[i]; nn = 0; } } } /*  * dump a block of lines, including numbers,stars: pr_align()  */ static dumpblock() dumpblock { register i; for(i =0; i < 2; i++) *po[i]−− = ‘\0’; ...dumpblock (void) putc(‘\n’, fx); for(i = 0; i < 2; i++) { if (*out[i] && (*out[i] != ‘ ’ || *(po[i]) != ‘’)) { if (i == 0) nums(i); if (i == 0 && *out[1]) stars(); putline(i);if (i == 0 && *out[1]) fprintf(fx, star); if (i == 1) nums(i); } } }/* * put out a number line: dumpblock()  */ static nums(ix) numsint  ix; /* index in out[] holding seq line */ { char nline[P_LINE];register i, j; register char *pn, *px, *py; for(pn = nline, i = 0; i <lmax+P_SPC; i++, pn++) *pn = ‘ ’; for (i = nc[ix], py = out[ix]; *py;py++, pn++) { if (*py == ‘ ’ || *py == ‘−’); *pn = ‘ ’; else { if (i%10== 0 || (i == 1 && nc[ix] != 1)) { j = (i < 0)? −i : i; for (px = pn; j;j /= 10, px−−) *px = j%10 + ‘0’; if (i < 0) *px = ‘−’; } else *pn = ‘ ’;i+ +; } } *pn = ‘\0’; nc[ix] = i; for (pn = nline; *pn; pn+ +) (void)putc(*pn, fx); (void) putc(‘\n’, fx); } /*  * put out a line (name,[num], seq, [num]): dumpblock()  */ static putline(ix) putline int   ix;{ ...putline int i; register char *px; for (px = namex[ix], i = 0; *px&& *px != ‘:’; px++, i++) (void) putc(*px, fx); for (;i < lmax + P_SPC;i++) (void) putc(‘ ’, fx); /* these count from 1:  * ni[] is currentelement (from 1)  * nc[] is number at start of current line  */ for (px= out[ix]; *px; px+ +) (void) putc(*px&0x7F, fx); (void) putc(‘\n’, fx);} /*  * put a line of stars (seqs always in out[0], out[1]): dumpblock() */ static stars() stars { int i; register char *p0, *p1, cx, *px; if(!*out[0] || (*out[0] == ‘ ’ && *(p0[0]) == ‘ ’) ||  !*out[1] ||(*out[1] == ‘ ’ && *(po[1]) == ‘ ’)) return; px = star; for (i = lmax +P_ SPC; i; i−−) *px++ = ‘ ’; for (p0 = out[0], p1 = out[1]; *p0 && *p1;p0++, p1++) { if (isalpha(*p0) && isalpha(*p1)) { if(xbm[*p0−‘A’]&xbm[*p1−‘A’]) { cx = ‘*’; nm+ +; } else if (!dna &&_day[*p0− ‘A’][*p1−‘A’] > 0) cx = ‘.’; else cx = ‘ ’; } else cx = ‘ ’;*px++ = cx; } *px++ = ‘\n’; *px = ‘\0’; } /*  * strip path or prefixfrom pn, return len: pr_align()  */ static stripname(pn) stripname char*pn; /* file name (may be path) */ { register char *px, *py; py = 0; for(px = pn; *px; px++) if (*px == ‘/’) py = px + 1; if (py) (void)strcpy(pn, py); return(strlen(pn)); } /*  * cleanup() -- cleanup any tmpfile  * getseq() -- read in seq, set dna, len, maxlen  * g_calloc() --calloc() with error checkin  * readjmps() -- get the good jmps, from tmpfile if necessary  * writejmps() -- write a filled array of jmps to atmp file: nw()  */ #include “nw.h” #include <sys/file.h> char *jname =“/tmp/homgXXXXXX”; /* tmp file for jmps */ FILE *fj; int cleanup(); /*cleanup tmp file */ long lseek(); /*  * remove any tmp file if we blow */ cleanup(i) cleanup int i; { if (fj) (void) unlink(jname); exit(i); }/*  * read, return ptr to seq, set dna, len, maxlen  * skip linesstarting with ‘;’, ‘<’, or ‘>’  * seq in upper or lower case  */ char *getseq(file, len) getseq char *file; /* file name */ int *len; /* seqlen */ { char line[1024], *pseq; register char *px, *py; int natgc,tlen; FILE *fp; if ((fp = fopen(file, “r”)) == 0) { fprintf(stderr, “%s:can't read %s\n”, prog, file); exit(1); } tlen = natgc = 0; while(fgets(line, 1024, fp)) { if (*line == ‘;’ || *line == ‘<’ || *line ==‘>’) continue; for (px = line; *px != ‘\n’; px+ +) if (isupper(*px) ||islower(*px)) tlen++; } if ((pseq = malloc((unsigned)(tlen+ 6))) == 0) {fprintf(stderr, “%s: malloc() failed to get %d bytes for %s\n”, prog,tlen+6, file); exit(1); } pseq[0] = pseq[1] = pseq[2] = pseq[3] = ‘\0’;...getseq py = pseq + 4; *len = tlen; rewind(fp); while (fgets(line,1024, fp)) { if (*line == ‘;’ || *line == ‘<’ || *line == ‘>’) continue;for (px = line; *px != ‘\n’; px++) { if (isupper(*px)) *py++ = *px; elseif (islower(*px)) *py+ + = toupper(*px); if (index(“ATGCU” , *(py−1)))natgc+ +; } } *py++ = ‘\0’; *py = ‘\0’; (void) fclose(fp); dna = natgc >(tlen/3); return(pseq+4); } char * g_calloc(msg, nx, sz) g_calloc char*msg; /* program, calling routine */ int nx, sz; /* number and size ofelements */ { char *px, *calloc(); if ((px = calloc((unsigned)nx,(unsigned)sz)) == 0) { if (*msg) { fprintf(stderr, “%s: g_calloc()failed %s (n= %d, sz= %d)\n”, prog, msg, nx, sz); exit(1); } }return(px); } /*  * get final jmps from dx[] or tmp file, set pp[],reset dmax: main()  */ readjmps() readjmps { int fd = −1; int siz, i0,i1; register i, j, xx; if (fj) { (void) fclose(fj); if ((fd =open(jname, O_RDONLY, 0)) < 0) { fprintf(stderr, “%s: can't open()%s\n”, prog, jname); cleanup(1); } } for (i = i0 = i1 = 0, dmax0 = dmax,xx = len0; ; i++) { while (1) { for (j = dx[dmax].ijmp; j >= 0 &&dx[dmax].jp.x[j] >= xx; j−−) ...readjmps if (j < 0 && dx[dmax].offset &&fj) { (void) lseek(fd, dx[dmax].offset, 0); (void) read(fd, (char*)&dx[dmax].jp, sizeof(struct jmp)); (void) read(fd, (char*)&dx[dmax].offset, sizeof(dx[dmax].offset)); dx[dmax].ijmp = MAXJMP−1;} else break; } if (i > = JMPS) { fprintf(stderr, “%s: too many gaps inalignment\n”, prog); cleanup(1); } if (j >= 0) { siz = dx[dmax].jp.n[j];xx = dx[dmax].jp.x[j]; dmax += siz; if (siz < 0) { /* gap in second seq*/ pp[1].n[il] = −siz; xx += siz; /* id = xx − yy + len1 − 1  */pp[1].x[il] = xx − dmax + len1 − 1; gapy+ +; ngapy −= siz; /* ignoreMAXGAP when doing endgaps */ siz = (−siz < MAXGAP || endgaps)? −siz :MAXGAP; il++; } else if (siz > 0) { /* gap in first seq */ pp[0].n[i0] =siz; pp[0].x[i0] = xx; gapx+ +; ngapx += siz; /* ignore MAXGAP whendoing endgaps */ siz = (siz < MAXGAP || endgaps)? siz : MAXGAP; i0+ +; }} else break; } /* reverse the order of jmps  */ for (j = 0, i0−−; j <i0; j++, i0−−) { i = pp[0].n[j]; pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] =i; i = pp[0].x[j]; pp[0].x[j] = pp[0].x[i0]; pp[0].x[i0] = i; } for (j =0, i1−−; j < i1; j++, i1−−) { i = pp[1].n[j]; pp[1].n[j] = pp[1].n[i1];pp[1].n[i1] = i; i = pp[1].x[j]; pp[1].x[j] = pp[1].x[i1]; pp[1].x[i1] =i; } if (fd > = 0) (void) close(fd); if (fj) { (void) unlink(jname); fj= 0; offset = 0; } } /*  * write a filled jmp struct offset of the prevone (if any): nw()  */ writejmps(ix) writejmps int ix; { char *mktemp();if (!fj) { if (mktemp(jname) < 0) { fprintf(stderr, “%s: can't mktemp()%s\n”, prog, jname); cleanup(1); } if ((fj = fopen(jname, “w”) == 0) {fprintf(stderr, “%s: can't write %s\n”, prog, jname); exit(1); } }(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj); (void)fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj); }

TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 15 = 33.3%

TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 10 = 50%

TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) ComparisonNNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA % nucleic acid sequenceidentity = (the number of identically matching nucleotides between thetwo nucleic acid sequences as determined by ALIGN-2) divided by (thetotal number of nucleotides of the PRO-DNA nucleic acid sequence) = 6divided by 14 = 42.9%

TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNANNNNLLLVV (Length = 9 nucleotides) % nucleic acid sequence identity =(the number of identically matching nucleotides between the two nucleicacid sequences as determined by ALIGN-2) divided by (the total number ofnucleotides of the PRO-DNA nucleic acid sequence) = 4 divided by 12 =33.3%II. Compositions and Methods of the Invention

A. Full-length PRO Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO polypeptides. In particular, cDNAs encoding various PROpolypeptides have been identified and isolated, as disclosed in furtherdetail in the Examples below. It is noted that proteins produced inseparate expression rounds may be given different PRO numbers but theUNQ number is unique for any given DNA and the encoded protein, and willnot be changed. However, for sake of simplicity, in the presentspecification the protein encoded by the full length native nucleic acidmolecules disclosed herein as well as all further native homologues andvariants included in the foregoing definition of PRO, will be referredto as “PRO/number”, regardless of their origin or mode of preparation.

As disclosed in the Examples below, various cDNA clones have beendeposited with the ATCC. The actual nucleotide sequences of those clonescan readily be determined by the skilled artisan by sequencing of thedeposited clone using routine methods in the art. The predicted aminoacid sequence can be determined from the nucleotide sequence usingroutine skill. For the PRO polypeptides and encoding nucleic acidsdescribed herein, Applicants have identified what is believed to be thereading frame best identifiable with the sequence information availableat the time.

1. Full-length PRO1800 Polypeptides

Using the WU-BLAST2 sequence alignment computer program, it has beenfound that a portion of the full-length native sequence PRO1800 (shownin FIG. 2 and SEQ ID NO:2) has certain amino acid sequence identity withthe human Hep27 protein (HE27_HUMAN). Accordingly, it is presentlybelieved that PRO1800 disclosed in the present application is a newlyidentified Hep27 homolog and possesses activity typical of that protein.

2. Full-length PRO539 Polypeptides

Using the WU-BLAST2 sequence alignment computer program, it has beenfound that a portion of the full-length native sequence PRO539 (shown inFIG. 4 and SEQ ID NO:7) has certain amino acid sequence identity with aportion of a kinesin-related protein from Drosophila melanogaster(AF019250_(—)1). Accordingly, it is presently believed that PRO539disclosed in the present application is a newly identified member of theHedgehog signaling pathway protein family and possesses activity typicalof the Drosophila Costal-2 protein.

3. Full-length PRO982 Polypeptides

As far as is known, the DNA57700-1408 sequence encodes a novel secretedfactor designated herein as PRO982. Although, using WU-BLAST2 sequencealignment computer programs, some sequence identities with knownproteins were revealed.

4. Full-length PRO1434 Polypeptides

Using the WU-BLAST2 sequence alignment computer program, it has beenfound that a portion of the full-length native sequence PRO1434 (shownin FIG. 8 and SEQ ID NO:11) has certain amino acid sequence identitywith the mouse nel protein precursor (NEL_MOUSE). Accordingly, it ispresently believed that PRO1434 disclosed in the present application isa newly identified nel homolog and may possess activity typical of thenel protein family.

5. Full-length PRO1863 Polypeptides

The DNA59847-2510 clone was isolated from a human prostate tissuelibrary. As far as is known, the DNA59847-2510 sequence encodes a novelfactor designated herein as PRO1863; using the WU-BLAST2 sequencealignment computer program, no significant sequence identities to anyknown proteins were revealed.

6. Full-length PRO1917 Polypeptides Using WU-BLAST2 sequence alignmentcomputer programs, it has been found that amino acids 41 to 487 ofPRO1917 (shown in FIG. 12 and SEQ ID NO:18) has certain amino acidsequence identity with an inositol phosphatase designated in the Dayhoffdatabase as “AF012714_(—)1”. Accordingly, it is presently believed thatPRO1917 disclosed in the present application is a newly identifiedmember of inositol phosphatase family and may possess enzymatic activitytypical of inositol phosphatases.

7. Full-length PRO1868 Polypeptides

Using the WU-BLAST2 sequence alignment computer program, it has beenfound that a portion of the full-length native sequence PRO1868 (shownin FIG. 14 and SEQ ID NO:20) has certain amino acid sequence identitywith the human A33 antigen protein (P_W14146). Accordingly, it ispresently believed that PRO1868 disclosed in the present application isa newly identified A33 antigen homolog which may possess activity and/orexpression patterns typical of the A33 antigen protein. The PRO1868polypeptide may find use in the therapeutic treatment of inflammatorydiseases as described above and colorectal cancer.

8. Full-length PRO3434 Polypeptides

The DNA77631-2537 clone was isolated from a human aortic tissue libraryusing a trapping technique that selects for nucleotide sequencesencoding secreted proteins. As far as is known, the DNA77631-2537sequence encodes a novel factor designated herein as PRO3434; using theWU-BLAST2 sequence alignment computer program, no significant sequenceidentities to any known proteins were revealed.

9. Full-length PRO1927 Polypeptides

Using WU-BLAST2 sequence alignment computer programs, it has been foundthat a full-length native sequence PRO1927 (FIG. 18; SEQ ID NO:24) hascertain amino acid sequence identity with the amino acid sequence of theprotein designated “AB000628_(—)1” in the Dayhoff database. Accordingly,it is presently believed that PRO1927 disclosed in the presentapplication is a newly identified member of the glycosyltransferasefamily of proteins and may possess glycosylation activity.

B. PRO Polypeptide Variants

In addition to the full-length native sequence PRO polypeptidesdescribed herein, it is contemplated that PRO variants can be prepared.PRO variants can be prepared by introducing appropriate nucleotidechanges into the PRO DNA, and/or by synthesis of the desired PROpolypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the PRO, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

Variations in the native full-length sequence PRO or in various domainsof the PRO described herein, can be made, for example, using any of thetechniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934.Variations may be a substitution, deletion or insertion of one or morecodons encoding the PRO that results in a change in the amino acidsequence of the PRO as compared with the native sequence PRO. Optionallythe variation is by substitution of at least one amino acid with anyother amino acid in one or more of the domains of the PRO. Guidance indetermining which amino acid residue may be inserted, substituted ordeleted without adversely affecting the desired activity may be found bycomparing the sequence of the PRO with that of homologous known proteinmolecules and minimizing the number of amino acid sequence changes madein regions of high homology. Amino acid substitutions can be the resultof replacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

PRO polypeptide fragments are provided herein. Such fragments may betruncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the PRO polypeptide.

PRO fragments may be prepared by any of a number of conventionaltechniques. Desired peptide fragments may be chemically synthesized. Analternative approach involves generating PRO fragments by enzymaticdigestion, e.g., by treating the protein with an enzyme known to cleaveproteins at sites defined by particular amino acid residues, or bydigesting the DNA with suitable restriction enzymes and isolating thedesired fragment. Yet another suitable technique involves isolating andamplifying a DNA fragment encoding a desired polypeptide fragment, bypolymerase chain reaction (PCR). Oligonucleotides that define thedesired termini of the DNA fragment are employed at the 5′ and 3′primers in the PCR. Preferably, PRO polypeptide fragments share at leastone biological and/or immunological activity with the native PROpolypeptide disclosed herein.

In particular embodiments, conservative substitutions of interest areshown in Table 1 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 6 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gin (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T)ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; leu ala; norleucine

Substantial modifications in functionor immunologicalidentity of the PROpolypeptide are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

-   (1) hydrophobic: norleucine, met, ala, val, leu, ile;-   (2) neutral hydrophilic: cys, ser, thr;-   (3) acidic: asp, glu;-   (4) basic: asn, gin, his, lys, arg;-   (5) residues that influence chain orientation: gly, pro; and-   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Such substituted residues also may be introduced into the conservativesubstitution sites or, more preferably, into the remaining(non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

C. Modifications of PRO

Covalent modifications of PRO are included within the scope of thisinvention. One type of covalent modification includes reacting targetedamino acid residues of a PRO polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or the N- orC- terminal residues of the PRO. Derivatization with bifunctional agentsis useful, for instance, for crosslinking PRO to a water-insolublesupport matrix or surface for use in the method for purifying anti-PROantibodies, and vice-versa. Commonly used crosslinking agents include,e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties. W. H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the PRO polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence PRO (eitherby removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequencePRO. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present.

Addition of glycosylation sites to the PRO polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence PRO (for O-linkedglycosylation sites). The PRO amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the PRO polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thePRO polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published Sep. 11, 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the PRO polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of PRO comprises linking the PROpolypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

The PRO of the present invention may also be modified in a way to form achimeric molecule comprising PRO fused to another, heterologouspolypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of thePRO with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the PRO. The presence ofsuch epitope-tagged forms of the PRO can be detected using an antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe PRO to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA.87:6393-6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of the PRO with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a PRO polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

D. Preparation of PRO

The description below relates primarily to production of PRO byculturing cells transformed or transfected with a vector containing PROnucleic acid. It is, of course, contemplated that alternative methods,which are well known in the art, may be employed to prepare PRO. Forinstance, the PRO sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al., Solid-Phase Peptide Synthesis, W. H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of thePRO may be chemically synthesized separately and combined using chemicalor enzymatic methods to produce the full-length PRO.

1. Isolation of DNA Encoding PRO

DNA encoding PRO may be obtained from a cDNA library prepared fromtissue believed to possess the PRO mRNA and to express it at adetectable level. Accordingly, human PRO DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The PRO-encoding gene may also be obtainedfrom a genomic library or by known synthetic procedures (e.g., automatednucleic acid synthesis).

Libraries can be screened with probes (such as antibodies to the PRO oroligonucleotides of at least about 20-80 bases) designed to identify thegene of interest or the protein encoded by it. Screening the cDNA orgenomic library with the selected probe may be conducted using standardprocedures, such as described in Sambrook et al., Molecular Cloning: ALaboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).An alternative means to isolate the gene encoding PRO is to use PCRmethodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for PRO production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.The culture conditions, such as media, temperature, pH and the like, canbe selected by the skilled artisan without undue experimentation. Ingeneral, principles, protocols, and practical techniques for maximizingthe productivity of cell cultures can be found in Mammalian CellBiotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991)and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published Jun. 29, 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tona ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvG kan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued Aug. 7, 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for PRO-encodingvectors. Saccharomyces cerevisiae is a commonly used lower eukaryotichost microorganism. Others include Schizosaccharomyces pombe (Beach andNurse, Nature, 290: 140 [1981]; EP 139,383 published May 2, 1985);Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742[1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278[1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 publishedOct. 31, 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published Jan. 10, 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284-289 [1983]; Tilbum et al., Gene,26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated PRO are derivedfrom multicellular organisms. Examples of invertebrate cells includeinsect cells such as Drosophila S2 and Spodoptera Sf9, as well as plantcells. Examples of useful mammalian host cell lines include Chinesehamster ovary (CHO) and COS cells. More specific examples include monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinesehamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad.Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCCCCL51). The selection of the appropriate host cell is deemed to bewithin the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

The PRO may be produced recombinantly not only directly, but also as afusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe PRO-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990), orthe signal described in WO 90/13646 published Nov. 15, 1990. Inmammalian cell expression, mammalian signal sequences may be used todirect secretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up thePRO-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the PRO-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg. 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphateisomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

PRO transcription from vectors in mammalian host cells is controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding the PRO by higher eukaryotes may beincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp, thatact on a promoter to increase its transcription. Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO coding sequence, but is preferably located at a site 5′ from thepromoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding PRO.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of PRO in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to PRO DNAand encoding a specific antibody epitope.

5. Purification of Polypeptide

Forms of PRO may be recovered from culture medium or from host celllysates. If membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of PRO can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

It may be desired to purify PRO from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of thePRO. Various methods of protein purification may be employed and suchmethods are known in the art and described for example in Deutscher,Methods in Enzymology 182 (1990); Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York (1982). Thepurification step(s) selected will depend, for example, on the nature ofthe production process used and the particular PRO produced.

E. Uses for PRO

Nucleotide sequences (or their complement) encoding PRO have variousapplications in the art of molecular biology, including uses ashybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. PRO nucleic acid will also beuseful for the preparation of PRO polypeptides by the recombinanttechniques described herein.

The full-length native sequence PRO gene, or portions thereof, may beused as hybridization probes for a cDNA library to isolate thefull-length PRO cDNA or to isolate still other cDNAs (for instance,those encoding naturally-occurring variants of PRO or PRO from otherspecies) which have a desired sequence identity to the native PROsequence disclosed herein. Optionally, the length of the probes will beabout 20 to about 50 bases. The hybridization probes may be derived fromat least partially novel regions of the full length native nucleotidesequence wherein those regions may be determined without undueexperimentation or from genomic sequences including promoters, enhancerelements and introns of native sequence PRO. By way of example, ascreening method will comprise isolating the coding region of the PROgene using the known DNA sequence to synthesize a selected probe ofabout 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or ³⁵S, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the PRO gene of the present invention can beused to screen libraries of human cDNA, genomic DNA or mRNA to determinewhich members of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below.

Any EST sequences disclosed in the present application may similarly beemployed as probes, using the methods disclosed herein.

Other useful fragments of the PRO nucleic acids include antisense orsense oligonucleotides comprising a singe-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target PRO mRNA (sense) or PRODNA (antisense) sequences. Antisense or sense oligonucleotides,according to the present invention, comprise a fragment of the codingregion of PRO DNA. Such a fragment generally comprises at least about 14nucleotides, preferably from about 14 to 30 nucleotides. The ability toderive an antisense or a sense oligonucleotide, based upon a cDNAsequence encoding a given protein is described in, for example, Steinand Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al.(BioTechniques 6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of PRO proteins.Antisense or sense oligonucleotides further comprise oligonucleotideshaving modified sugar-phosphodiester backbones (or other sugar linkages,such as those described in WO 91/06629) and wherein such sugar linkagesare resistant to endogenous nucleases. Such oligonucleotides withresistant sugar linkages are stable in vivo (i.e., capable of resistingenzymatic degradation) but retain sequence specificity to be able tobind to target nucleotide sequences.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10048, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

Antisense or sense RNA or DNA molecules are generally at least about 5bases in length, about 10 bases in length, about 15 bases in length,about 20 bases in length, about 25 bases in length, about 30 bases inlength, about 35 bases in length, about 40 bases in length, about 45bases in length, about 50 bases in length, about 55 bases in length,about 60 bases in length, about 65 bases in length, about 70 bases inlength, about 75 bases in length, about 80 bases in length, about 85bases in length, about 90 bases in length, about 95 bases in length,about 100 bases in length, or more.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related PRO coding sequences.

Nucleotide sequences encoding a PRO can also be used to constructhybridization probes for mapping the gene which encodes that PRO and forthe genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

When the coding sequences for PRO encode a protein which binds toanother protein (example, where the PRO is a receptor), the PRO can beused in assays to identify the other proteins or molecules involved inthe binding interaction. By such methods, inhibitors of thereceptor/ligand binding interaction can be identified. Proteins involvedin such binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction. Also,the receptor PRO can be used to isolate correlative ligand(s). Screeningassays can be designed to find lead compounds that mimic the biologicalactivity of a native PRO or a receptor for PRO. Such screening assayswill include assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates. Small molecules contemplated include syntheticorganic or inorganic compounds. The assays can be performed in a varietyof formats, including protein-protein binding assays, biochemicalscreening assays, immunoassays and cell based assays, which are wellcharacterized in the art.

Nucleic acids which encode PRO or its modified forms can also be used togenerate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding PRO can be used to clone genomic DNA encodingPRO in accordance with established techniques and the genomic sequencesused to generate transgenic animals that contain cells which express DNAencoding PRO. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for PRO transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding PRO introduced into the germ lineof the animal at an embryonic stage can be used to examine the effect ofincreased expression of DNA encoding PRO. Such animals can be used astester animals for reagents thought to confer protection from, forexample, pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of PRO can be used to construct aPRO “knock out” animal which has a defective or altered gene encodingPRO as a result of homologous recombination between the endogenous geneencoding PRO and altered genomic DNA encoding PRO introduced into anembryonic stem cell of the animal. For example, cDNA encoding PRO can beused to clone genomic DNA encoding PRO in accordance with establishedtechniques. A portion of the genomic DNA encoding PRO can be deleted orreplaced with another gene, such as a gene encoding a selectable markerwhich can be used to monitor integration. Typically, several kilobasesof unaltered flanking DNA (both at the 5′ and 3′ ends) are included inthe vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for adescription of homologous recombination vectors]. The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced DNA has homologously recombined withthe endogenous DNA are selected [see e.g., Li et al., Cell, 69:915(1992)]. The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse or rat) to form aggregation chimeras [see e.g.,Bradley, in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term to create a “knockout” animal. Progeny harboring the homologously recombined DNA in theirgerm cells can be identified by standard techniques and used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA. Knockout animals can be characterized for instance, fortheir ability to defend against certain pathological conditions and fortheir development of pathological conditions due to absence of the PROpolypeptide.

Nucleic acid encoding the PRO polypeptides may also be used in genetherapy. In gene therapy applications, genes are introduced into cellsin order to achieve in vivo synthesis of a therapeutically effectivegenetic product, for example for replacement of a defective gene. “Genetherapy” includes both conventional gene therapy where a lasting effectis achieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83:4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, -4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

The PRO polypeptides described herein may also be employed as molecularweight markers for protein electrophoresis purposes and the isolatednucleic acid sequences may be used for recombinantly expressing thosemarkers.

The nucleic acid molecules encoding the PRO polypeptides or fragmentsthereof described herein are useful for chromosome identification. Inthis regard, there exists an ongoing need to identify new chromosomemarkers, since relatively few chromosome marking reagents, based uponactual sequence data are presently available. Each PRO nucleic acidmolecule of the present invention can be used as a chromosome marker.

The PRO polypeptides and nucleic acid molecules of the present inventionmay also be used for tissue typing, wherein the PRO polypeptides of thepresent invention may be differentially expressed in one tissue ascompared to another. PRO nucleic acid molecules will find use forgenerating probes for PCR, Northern analysis, Southern analysis andWestern analysis.

The PRO polypeptides described herein may also be employed astherapeutic agents. The PRO polypeptides of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the PRO product hereof is combined in admixturewith a pharmaceutically acceptable carrier vehicle. Therapeuticformulations are prepared for storage by mixing the active ingredienthaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrateand other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, or dextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as TWEEN™,PLURONICS™ or PEG.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42-96.

When in vivo administration of a PRO polypeptide or agonist orantagonist thereof is employed, normal dosage amounts may vary fromabout 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day,preferably about 1 μg/kg/day to 10 mg/kg/day, depending upon the routeof administration. Guidance as to particular dosages and methods ofdelivery is provided in the literature; see, for example, U.S. Pat. Nos.4,657,760; 5,206,344; or 5,225,212. It is anticipated that differentformulations will be effective for different treatment compounds anddifferent disorders, that administration targeting one organ or tissue,for example, may necessitate delivery in a manner different from that toanother organ or tissue.

Where sustained-release administration of a PRO polypeptide is desiredin a formulation with release characteristics suitable for the treatmentof any disease or disorder requiring administration of the PROpolypeptide, microencapsulation of the PRO polypeptide is contemplated.Microencapsulation of recombinant proteins for sustained release hasbeen successfully performed with human growth hormone (rhGH),interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Horaet al., Bio/Technology, 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010.

The sustained-release formulations of these proteins were developedusing poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp. 1-41.

This invention encompasses methods of screening compounds to identifythose that mimic the PRO polypeptide (agonists) or prevent the effect ofthe PRO polypeptide (antagonists). Screening assays for antagonist drugcandidates are designed to identify compounds that bind or complex withthe PRO polypeptides encoded by the genes identified herein, orotherwise interfere with the interaction of the encoded polypeptideswith other cellular proteins. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

All assays for antagonists are common in that they call for contactingthe drug candidate with a PRO polypeptide encoded by a nucleic acididentified herein under conditions and for a time sufficient to allowthese two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the PRO polypeptide encoded by the gene identified herein orthe drug candidate is immobilized on a solid phase, e.g., on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the PRO polypeptide and drying. Alternatively, animmobilized antibody, e.g., a monoclonal antibody, specific for the PROpolypeptide to be immobilized can be used to anchor it to a solidsurface. The assay is performed by adding the non-immobilized component,which may be labeled by a detectable label, to the immobilizedcomponent, e.g., the coated surface containing the anchored component.When the reaction is complete, the non-reacted components are removed,e.g., by washing, and complexes anchored on the solid surface aredetected. When the originally non-immobilized component carries adetectable label, the detection of label immobilized on the surfaceindicates that complexing occurred. Where the originally non-immobilizedcomponent does not carry a label, complexing can be detected, forexample, by using a labeled antibody specifically binding theimmobilized complex.

If the candidate compound interacts with but does not bind to aparticular PRO polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340:245-246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a gene encoding a PROpolypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

To assay for antagonists, the PRO polypeptide may be added to a cellalong with the compound to be screened for a particular activity and theability of the compound to inhibit the activity of interest in thepresence of the PRO polypeptide indicates that the compound is anantagonist to the PRO polypeptide. Alternatively, antagonists may bedetected by combining the PRO polypeptide and a potential antagonistwith membrane-bound PRO polypeptide receptors or recombinant receptorsunder appropriate conditions for a competitive inhibition assay. The PROpolypeptide can be labeled, such as by radioactivity, such that thenumber of PRO polypeptide molecules bound to the receptor can be used todetermine the effectiveness of the potential antagonist. The geneencoding the receptor can be identified by numerous methods known tothose of skill in the art, for example, ligand panning and FACS sorting.Coligan et al., Current Protocols in Immun. 1(2): Chapter 5 (1991).Preferably, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the PRO polypeptide and a cDNAlibrary created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to the PROpolypeptide. Transfected cells that are grown on glass slides areexposed to labeled PRO polypeptide. The PRO polypeptide can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase. Following fixation andincubation, the slides are subjected to autoradiographic analysis.Positive pools are identified and sub-pools are prepared andre-transfected using an interactive sub-pooling and re-screeningprocess, eventually yielding a single clone that encodes the putativereceptor.

As an alternative approach for receptor identification, labeled PROpolypeptide can be photoaffinity-linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining the receptor can be excised, resolved into peptide fragments,and subjected to protein micro-sequencing. The amino acid sequenceobtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeled PROpolypeptide in the presence of the candidate compound. The ability ofthe compound to enhance or block this interaction could then bemeasured.

More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with PROpolypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of the PROpolypeptide that recognizes the receptor but imparts no effect, therebycompetitively inhibiting the action of the PRO polypeptide.

Another potential PRO polypeptide antagonist is an antisense RNA or DNAconstruct prepared using antisense technology, where, e.g., an antisenseRNA or DNA molecule acts to block directly the translation of mRNA byhybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature PRO polypeptides herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the PRO polypeptide. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into the PRO polypeptide (antisense—Okano, Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression(CRC Press: Boca Raton, Fla., 1988). The oligonucleotides describedabove can also be delivered to cells such that the antisense RNA or DNAmay be expressed in vivo to inhibit production of the PRO polypeptide.When antisense DNA is used, oligodeoxyribonucleotides derived from thetranslation-initiation site, e.g., between about −10 and +10 positionsof the target gene nucleotide sequence, are preferred.

Potential antagonists include small molecules that bind to the activesite, the receptor binding site, or growth factor or other relevantbinding site of the PRO polypeptide, thereby blocking the normalbiological activity of the PRO polypeptide. Examples of small moleculesinclude, but are not limited to, small peptides or peptide-likemolecules, preferably soluble peptides, and synthetic non-peptidylorganic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4:469471 (1994), and PCT publication No. WO 97/33551 (publishedSep. 18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

F. Anti-PRO Antibodies

The present invention further provides anti-PRO antibodies. Exemplaryantibodies include polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-PRO antibodies may comprise polyclonal antibodies. Methods ofpreparing polyclonal antibodies are known to the skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the PRO polypeptide or a fusion proteinthereof. It may be useful to conjugate the immunizing agent to a proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

The anti-PRO antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

The immunizing agent will typically include the PRO polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against PRO.Preferably, the binding specificity of monoclonal antibodies produced bythe hybridoma cells is determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). Such techniques and assays are known inthe art. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollard,Anal. Biochem. 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

3. Human and Humanized Antibodies

The anti-PRO antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe PRO, the other one is for any other antigen, and preferably for acell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., Science229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med.175:217-225 (1992) describe the production of a fully humanizedbispecific antibody F(ab′)₂ molecule. Each Fab′ fragment was separatelysecreted from E. coli and subjected to directed chemical coupling invitro to form the bispecific antibody. The bispecific antibody thusformed was able to bind to cells overexpressing the ErbB2 receptor andnormal human T cells, as well as trigger the lytic activity of humancytotoxic lymphocytes against human breast tumor targets.

Various technique for making and isolating bispecific antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bispecific antibodies have been produced using leucine zippers.Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipperpeptides from the Fos and Jun proteins were linked to the Fab′ portionsof two different antibodies by gene fusion. The antibody homodimers werereduced at the hinge region to form monomers and then re-oxidized toform the antibody heterodimers. This method can also be utilized for theproduction of antibody homodimers. The “diabody” technology described byHollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) hasprovided an alternative mechanism for making bispecific antibodyfragments. The fragments comprise a heavy-chain variable domain (V_(H))connected to a light-chain variable domain (V_(L)) by a linker which istoo short to allow pairing between the two domains on the same chain.Accordingly, the V_(H) and V_(L) domains of one fragment are forced topair with the complementary V_(L) and V_(H) domains of another fragment,thereby forming two antigen-binding sites. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (sFv) dimershas also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies may bind to two different epitopes on agiven PRO polypeptide herein. Alternatively, an anti-PRO polypeptide armmay be combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD 16) so as to focus cellular defense mechanisms to thecell expressing the particular PRO polypeptide. Bispecific antibodiesmay also be used to localize cytotoxic agents to cells which express aparticular PRO polypeptide. These antibodies possess a PRO-binding armand an arm which binds a cytotoxic agent or a radionuclide chelator,such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody ofinterest binds the PRO polypeptide and further binds tissue factor (TF).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection WO 91/00360; WO 92/200373; EP 030891.It is contemplated that the antibodies may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.Pat. No. 4,676,980.

6. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) may beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design. 3: 219-230 (1989).

7. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generationof such immunoconjugateshave been described above. Enzymatically active toxins and fragmentsthereof that can be used include diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is conjugatedto a cytotoxic agent (e.g., a radionucleotide).

8. Immunoliposomes

The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, andPEG-derivatizedphosphatidylethanolamine(PEG-PE). Liposomes are extrudedthrough filters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.81(19): 1484 (1989).

9. Pharmaceutical Compositions of Antibodies

Antibodies specifically binding a PRO polypeptide identified herein, aswell as other molecules identified by the screening assays disclosedhereinbefore, can be administered for the treatment of various disordersin the form of pharmaceutical compositions.

If the PRO polypeptide is intracellular and whole antibodies are used asinhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889-7893 (1993). The formulation herein may also containmore than one active compound as necessary for the particular indicationbeing treated, preferably those with complementary activities that donot adversely affect each other. Alternatively, or in addition, thecomposition may comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and yethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfideinterchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

G. Uses for anti-PRO Antibodies

The anti-PRO antibodies of the invention have various utilities. Forexample, anti-PRO antibodies may be used in diagnostic assays for PRO,e.g., detecting its expression in specific cells, tissues, or serum.Various diagnostic assay techniques known in the art may be used, suchas competitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Anti-PRO antibodies also are useful for the affinity purification of PROfrom recombinant cell culture or natural sources. In this process, theantibodies against PRO are immobilized on a suitable support, such aSephadex resin or filter paper, using methods well known in the art. Theimmobilized antibody then is contacted with a sample containing the PROto be purified, and thereafter the support is washed with a suitablesolvent that will remove substantially all the material in the sampleexcept the PRO, which is bound to the immobilized antibody. Finally, thesupport is washed with another suitable solvent that will release thePRO from the antibody.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example 1 Extracellular Domain Homology Screening to Identify NovelPolypeptides and cDNA Encoding Therefor

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public databases (e.g., Dayhoff, GenBank), andproprietary databases (e.g. LIFESEQ™, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST-2 (Altschul et al., Methods in Enzymology 266:460-480 (1996)) as acomparison of the ECD protein sequences to a 6 frame translation of theEST sequences. Those comparisons with a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into consensus DNA sequences with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.).

Using this extracellular domain homology screen, consensus DNA sequenceswere assembled relative to the other identified EST sequences usingphrap. In addition, the consensus DNA sequences obtained were often (butnot always) extended using repeated cycles of BLAST or BLAST-2 and phrapto extend the consensus sequence as far as possible using the sources ofEST sequences discussed above.

Based upon the consensus sequences obtained as described above,oligonucleotides were then synthesized and used to identify by PCR acDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for a PROpolypeptide. Forward and reverse PCR primers generally range from 20 to30 nucleotides and are often designed to give a PCR product of about100-1000 bp in length. The probe sequences are typically 40-55 bp inlength. In some cases, additional oligonucleotides are synthesized whenthe consensus sequence is greater than about 1-1.5 kbp. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, with the PCR primer pair. A positivelibrary was then used to isolate clones encoding the gene of interestusing the probe oligonucleotide and one of the primer pairs.

The cDNA libraries used to isolate the cDNA clones were constructed bystandard methods using commercially available reagents such as thosefrom Invitrogen, San Diego, Calif. The cDNA was primed with oligo dTcontaining a NotI site, linked with blunt to SalI hemikinased adaptors,cleaved with NotI, sized appropriately by gel electrophoresis, andcloned in a defined orientation into a suitable cloning vector (such aspRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain theSfiI site; see, Holmes et al., Science, 253:1278-1280 (1991)) in theunique XhoI and NotI sites.

Example 2 Isolation of cDNA clones by Amylase Screening

1. Preparation of Oligo dT Primed cDNA Library

mRNA was isolated from a human tissue of interest using reagents andprotocols from Invitrogen, San Diego, Calif. (Fast Track 2). This RNAwas used to generate an oligo dT primed cDNA library in the vector pRK5Dusing reagents and protocols from Life Technologies, Gaithersburg, Md.(Super Script Plasmid System). In this procedure, the double strandedcDNA was sized to greater than 1000 bp and the SalI/NotI linkered cDNAwas cloned into XhoI/NotI cleaved vector. pRK5D is a cloning vector thathas an sp6 transcription initiation site followed by an SfiI restrictionenzyme site preceding the XhoI/NotI cDNA cloning sites.

2. Preparation of Random Primed cDNA Library

A secondary cDNA library was generated in order to preferentiallyrepresent the 5′ ends of the primary cDNA clones. Sp6 RNA was generatedfrom the primary library (described above), and this RNA was used togenerate a random primed cDNA library in the vector pSST-AMY.0 usingreagents and protocols from Life Technologies (Super Script PlasmidSystem, referenced above). In this procedure the double stranded cDNAwas sized to 500-1000 bp, linkered with blunt to NotI adaptors, cleavedwith SfiI, and cloned into SfiI/NotI cleaved vector. pSST-AMY.0 is acloning vector that has a yeast alcohol dehydrogenase promoter precedingthe cDNA cloning sites and the mouse amylase sequence (the maturesequence without the secretion signal) followed by the yeast alcoholdebydrogenase terminator, after the cloning sites. Thus, cDNAs clonedinto this vector that are fused in frame with amylase sequence will leadto the secretion of amylase from appropriately transfected yeastcolonies.

3. Transformation and Detection

DNA from the library described in paragraph 2 above was chilled on iceto which was added electrocompetent DH10B bacteria (Life Technologies,20 ml). The bacteria and vector mixture was then electroporated asrecommended by the manufacturer. Subsequently, SOC media (LifeTechnologies, 1 ml) was added and the mixture was incubated at 37° C.for 30 minutes. The transformants were then plated onto 20 standard 150mm LB plates containing ampicillin and incubated for 16 hours (37° C.).Positive colonies were scraped off the plates and the DNA was isolatedfrom the bacterial pellet using standard protocols, e.g. CsCl-gradient.The purified DNA was then carried on to the yeast protocols below.

The yeast methods were divided into three categories: (1) Transformationof yeast with the plasmid/cDNA combined vector; (2) Detection andisolation of yeast clones secreting amylase; and (3) PCR amplificationof the insert directly from the yeast colony and purification of the DNAfor sequencing and further analysis.

The yeast strain used was HD56-5A (ATCC-90785). This strain has thefollowing genotype: MAT alpha, ura3-52, leu2-3, leu2-112, his3-11,his3-15, MAL⁺, SUC⁺, GAL⁺. Preferably, yeast mutants can be employedthat have deficient post-translational pathways. Such mutants may havetranslocation deficient alleles in sec71, sec72, sec62, with truncatedsec71 being most preferred. Alternatively, antagonists (includingantisense nucleotides and/or ligands) which interfere with the normaloperation of these genes, other proteins implicated in this posttranslation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p, TDJ1p orSSA1p-4p) or the complex formation of these proteins may also bepreferably employed in combination with the amylase-expressing yeast.

Transformation was performed based on the protocol outlined by Gietz etal., Nucl. Acid. Res., 20:1425 (1992). Transformed cells were theninoculated from agar into YEPD complex media broth (100 ml) and grownovernight at 30° C. The YEPD broth was prepared as described in Kaiseret al., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold SpringHarbor, N.Y., p. 207 (1994). The overnight culture was then diluted toabout 2×10⁶ cells/ml (approx. OD₆₀₀=0.1) into fresh YEPD broth (500 ml)and regrown to 1×10⁷ cells/ml (approx. OD₆₀₀=0.4-0.5).

The cells were then harvested and prepared for transformation bytransfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5minutes, the supernatant discarded, and then resuspended into sterilewater, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in aBeckman GS-6KR centrifuge. The supernatant was discarded and the cellswere subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTApH 7.5, 100 mM Li₂OOCCH₃), and resuspended into LiAc/TE (2.5 ml).

Transformation took place by mixing the prepared cells (100 μl) withfreshly denatured single stranded salmon testes DNA (Lofstrand Labs,Gaithersburg, Md.) and transforming DNA (1 μg, vol.<10 μl) in microfugetubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE (600μl, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mMLi₂OOCCH₃, pH 7.5) was added. This mixture was gently mixed andincubated at 30° C. while agitating for 30 minutes. The cells were thenheat shocked at 42° C. for 15 minutes, and the reaction vesselcentrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted andresuspended into TE (500 μl, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followedby recentrifugation. The cells were then diluted into TE (1 ml) andaliquots (200 μl) were spread onto the selective media previouslyprepared in 150 mm growth plates (VWR).

Alternatively, instead of multiple small reactions, the transformationwas performed using a single, large scale reaction, wherein reagentamounts were scaled up accordingly.

The selective media used was a synthetic complete dextrose agar lackinguracil (SCD-Ura) prepared as described in Kaiser et al., Methods inYeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., p.208-210 (1994). Transformants were grown at 30° C. for 2-3 days.

The detection of colonies secreting amylase was performed by includingred starch in the selective growth media. Starch was coupled to the reddye (Reactive Red-120, Sigma) as per the procedure described by Biely etal., Anal. Biochem., 172:176-179 (1988). The coupled starch wasincorporated into the SCD-Ura agar plates at a final concentration of0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0(50-100 mM final concentration).

The positive colonies were picked and streaked across fresh selectivemedia (onto 150 mm plates) in order to obtain well isolated andidentifiable single colonies. Well isolated single colonies positive foramylase secretion were detected by direct incorporation of red starchinto buffered SCD-Ura agar. Positive colonies were determined by theirability to break down starch resulting in a clear halo around thepositive colony visualized directly.

4. Isolation of DNA by PCR Amplification

When a positive colony was isolated, a portion of it was picked by atoothpick and diluted into sterile water (30 μl) in a 96 well plate. Atthis time, the positive colonies were either frozen and stored forsubsequent analysis or immediately amplified. An aliquot of cells (5 μl)was used as a template for the PCR reaction in a 25 μl volumecontaining: 0.5 μl Klentaq (Clontech, Palo Alto, Calif.); 4.0 μl 10 mMdNTP's (Perkin Elmer-Cetus); 2.5 μl Kentaq buffer (Clontech); 0.25 μlforward oligo 1; 0.25 μl reverse oligo 2; 12.5 μl distilled water. Thesequence of the forward oligonucleotide 1 was:

(SEQ ID NO:25) 5′-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3′The sequence of reverse oligonucleotide 2 was:

(SEQ ID NO:26) 5′-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3′

PCR was then perfoemed as follows:

a. Denature 92° C., 5 minutes b. 3 cycles of: Denature 92° C., 30seconds Anneal 59° C., 30 seconds Extend 72° C., 60 seconds c. 3 cyclesof: Denature 92° C., 30 seconds Anneal 57° C., 30 seconds Extend 72° C.,60 seconds d. 25 cycles of: Denature 92° C., 30 seconds Anneal 55° C.,30 seconds Extend 72° C., 60 seconds e. Hold  4° C.

The underlined regions of the oligonucleotides annealed to the ADHpromoter region and the amylase region, respectively, and amplified a307 bp region from vector pSST-AMY.0 when no insert was present.Typically, the first 18 nucleotides of the 5′ end of theseoligonucleotides contained annealing sites for the sequencing primers.Thus, the total product of the PCR reaction from an empty vector was 343bp. However, signal sequence-fused cDNA resulted in considerably longernucleotide sequences.

Following the PCR, an aliquot of the reaction (5 μl) was examined byagarose gel electrophoresis in a 1% agarose gel using a Tris-Borate-EDTA(TBE) buffering system as described by Sambrook et al., supra. Clonesresulting in a single strong PCR product larger than 400 bp were furtheranalyzed by DNA sequencing after purification with a 96 Qiaquick PCRclean-up column (Qiagen Inc., Chatsworth, Calif.).

Example 3 Isolation of cDNA Clones Using Signal Algorithm Analysis

Various polypeptide-encoding nucleic acid sequences were identified byapplying a proprietary signal sequence finding algorithm developed byGenentech, Inc. (South San Francisco, Calif.) upon ESTs as well asclustered and assembled EST fragments from public (e.g., GenBank) and/orprivate (LIFESEQ®, Incyte Pharmaceuticals, Inc., Palo Alto, Calif.)databases. The signal sequence algorithm computes a secretion signalscore based on the character of the DNA nucleotides surrounding thefirst and optionally the second methionine codon(s) (ATG) at the 5′-endof the sequence or sequence fragment under consideration. Thenucleotides following the first ATG must code for at least 35unambiguous amino acids without any stop codons. If the first ATG hasthe required amino acids, the second is not examined. If neither meetsthe requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals. Use of thisalgorithm resulted in the identification of numerouspolypeptide-encoding nucleic acid sequences.

Example 4 Isolation of cDNA clones Encoding Human PRO1800

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example I above. This consensus sequence isherein designated DNA30934. Based on the DNA30934 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO1800.

PCR primers (forward and reverse) were synthesized:

forward PCR primer (30934.f1) 5′-GCATAATGGATGTCACTGAGG-3′ (SEQ ID NO:3)reverse PCR primer (30934.r1) 5′-AGAACAATCCTGCTGAAAGCTAG-3′ (SEQ IDNO:4)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30934 sequence which had the followingnucleotide sequenceHybridization Probe (30934.p1)

-   5′-GAAACGAGGAGGCGGCTCAGTGGTGATCGTGTCTTCCATAGCAGCC-3′ (SEQ ID NO :5)

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue. DNA sequencing of the clones isolated as described abovegave the full-length DNA sequence for PRO1800 (designated herein asDNA35672-2508 [FIG. 1, SEQ ID NO:1]; and the derived protein sequencefor PRO1800.

The entire nucleotide sequence of DNA35672-2508 is shown in FIG. 1 (SEQID NO:1). Clone DNA35672-2508 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 36-38and ending at the stop codon at nucleotide positions 870-872 (FIG. 1).The predicted polypeptide precursor is 278 amino acids long (FIG. 2).The full-length PRO1800 protein shown in FIG. 2 has an estimatedmolecular weight of about 29,537 daltons and a pI of about 8.97.Analysis of the full-length PRO1800 sequence shown in FIG. 2 (SEQ IDNO:2) evidences the presence of the following: a signal peptide fromabout amino acid 1 to about amino acid 15, a potential N-glycosylationsite from about amino acid 183 to about amino acid 186, potentialN-myristolation sites from about amino acid 43 to about amino acid 48,from about amino acid 80 to about amino acid 85, from about amino acid191 to about amino acid 196, from about amino acid 213 to about aminoacid 218 and from about amino acid 272 to about amino acid 277 and amicrobodies C-terminal targeting signal from about amino acid 276 toabout amino acid 278. Clone DNA35672-2508 has been deposited with ATCCon Dec 15, 1998 and is assigned ATCC deposit no. 203538.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 2 (SEQ ID NO:2), evidenced significant homology betweenthe PRO1800 amino acid sequence and the following Dayhoff sequences:HE27_HUMAN, CELF36H9_(—)1, CEF54F3_(—)3, A69621, AP000007_(—)227,UCPA_ECOLI, F69868, Y4LA_RHISN, DHK2_STRVN and DHG1_BACME.

Example 5 Isolation of cDNA clones Encoding Human PRO539

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1. This consensus sequence is hereindesignated DNA41882. Based on the DNA41882 consensus sequence shown,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO539.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue. DNA sequencing of the clones isolated as described abovegave the full-length DNA sequence for PRO539 (designated herein asDNA47465-1561 [FIG. 3, SEQ ID NO:6]; and the derived protein sequencefor PRO539.

The entire nucleotide sequence of DNA47465-1561 is shown in FIG. 3 (SEQID NO:6). Clone DNA47465-1561 contains a single open reading frame withan apparent translational initiation site at nucleotide positions186-188 and ending at the stop codon at nucleotide positions 2676-2678(FIG. 3). The predicted polypeptide precursor is 830 amino acids long(FIG. 4). The full-length PRO539 protein shown in FIG. 4 has anestimated molecular weight of about 95,029 daltons and a pI of about8.26. Analysis of the full-length PRO539 sequence shown in FIG. 4 (SEQID NO:7) evidences the presence of the following: leucine zipper patternsequences from about amino acid 557 to about amino acid 578 and fromabout amino acid 794 to about amino acid 815, potential N-glycosylationsites from about amino acid 133 to about amino acid 136 and from aboutamino acid 383 to about amino acid 386 and a kinesin-related proteinKif-4 coiled coil domain from about amino acid 231 to about amino acid672. Clone DNA47465-1561 has been deposited with ATCC on Feb. 9, 1999and is assigned ATCC deposit no. 203661.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 4 (SEQ ID NO:7), evidenced homology between the PRO539amino acid sequence and the following Dayhoff sequences: AF019250_(—)1,KIF4_MOUSE, TRHY_HUMAN, A56514, G02520, MYSP_HUMAN, AF041382_(—)1,A45592, HS125H2_(—)1 and HS68O2_(—)2.

Example 6 Isolation of cDNA clones Encoding Human PRO982

Use of the signal sequence algorithm described in Example 3 aboveallowed identification of a single Incyte EST sequence designated hereinas Incyte EST cluster sequence no. 43715. This EST sequence was comparedto a variety of EST databases which included public EST databases (e.g.,GenBank) and a proprietary EST DNA database (LIFESEQ™, IncytePharmaceuticals, Palo Alto, Calif.) to identify existing homologies. Thehomology search was performed using the computer program BLAST or BLAST2(Altshul et al., Methods in Enzymology 266:460-480 (1996)). Thosecomparisons resulting in a BLAST score of 70 (or in some cases 90) orgreater that did not encode known proteins were clustered and assembledinto a consensus DNA sequence with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.). The consensus sequenceobtained therefrom is designated DNA56095.

In light of an observed sequence homology between DNA56095 and Merck ESTno. AA024389, Merck EST clone AA024389 was obtained and sequenced. Thesequence, designated DNA57700-1408 (SEQ ID NO:8), is shown in FIG. 5. Itis the full-length DNA sequence for PRO982.

The full length clone shown in FIG. 5 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 26-28 and ending at the stop codon found at nucleotidepositions 401-403 (SEQ ID NO:8). The predicted polypeptide precursor is125 amino acids long, has a calculated molecular weight of approximately14,198 daltons and an estimated pI of approximately 9.01. Analysis ofthe full-length PRO982 sequence shown in FIG. 6 (SEQ ID NO:9) evidencesthe presence of a signal peptide from about amino acid 1 to about aminoacid 21 and potential anaphylatoxin domain from about amino acid 50 toabout amino acid 59. An analysis of the Dayhoff database (version 35.45SwissProt 35) evidenced homology between the PRO982 amino acid sequenceand the following Dayhoff sequences: RNTMDCV_(—)1; A48151; WAP_RAT;S24596; A53640; MT4_HUMAN; U93486_(—)1; SYNBILGFG_(—)1; P_R49917; andP_R41880. Clone DNA57700-1408 was deposited with the ATCC on Jan. 12,1999 and is assigned ATCC deposit no. 203583.

Example 7 Isolation of cDNA clones Encoding Human PRO1434

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example I above. This consensus sequence isherein designated DNA54187. Based on the DNA54187 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO1434.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-GAGGTGTCGCTGTGAAGCCAACGG-3′ (SEQ ID NO:12) reversePCR primer 5′-CGCTCGATTCTCCATGTGCCTTCC-3′ (SEQ ID NO:13)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA54187 sequence which had the followingnucleotide sequenceHybridization Probe

-   5′-GACGGAGTGTGTGGACCCTGTGTACGAGCCTGATCAGTGCTGTCC-3′ (SEQ ID NO:14)

RNA for construction of the cDNA libraries was isolated from humanretina tissue (LIB94). DNA sequencing of the clones isolated asdescribed above gave the full-length DNA sequence for PRO1434(designated herein as DNA68818-2536 [FIG. 7, SEQ ID NO:10]; and thederived protein sequence for PRO1434.

The entire nucleotide sequence of DNA68818-2536 is shown in FIG. 7 (SEQID NO:10). Clone DNA68818-2536 contains a single open reading frame withan apparent translational initiation site at nucleotide positions581-583 and ending at the stop codon at nucleotide positions 1556-1558(FIG. 7). The predicted polypeptide precursor is 325 amino acids long(FIG. 8). The full-length PRO1434 protein shown in FIG. 8 has anestimated molecular weight of about 35,296 daltons and a pI of about5.37. Analysis of the full-length PRO1434 sequence shown in FIG. 8 (SEQID NO:11) evidences the presence of a variety of important proteindomains as shown in FIG. 8. Clone DNA68818-2536 has been deposited withATCC on Feb. 9, 1999 and is assigned ATCC deposit no. 203657.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 8 (SEQ ID NO:11), evidenced significant homology betweenthe PRO1434 amino acid sequence and the following Dayhoff sequences:NEL_MOUSE, APMU_PIG, P_W37501,NEL_RAT, TSP1_CHICK, P_W37500,NEL2_HUMAN,MMU010792_(—)1, D86983_(—)1 and 10 MUCS_BOVIN.

Example 8 Isolation of cDNA clones Encoding Human PRO1863

Use of the signal sequence algorithm described in Example 3 aboveallowed identification of an EST cluster sequence from the Incytedatabase, designated Incyte EST cluster sequence no. 82468. This ESTcluster sequence was then compared to a variety of expressed sequencetag (EST) databases which included public EST databases (e.g., GenBank)and a proprietary EST DNA database (Lifeseq®, Incyte Pharmaceuticals,Palo Alto, Calif.) to identify existing homologies. The homology searchwas performed using the computer program BLAST or BLAST2 (Altshul etal., Methods in Enzymology 266:460480 (1996)). Those comparisonsresulting in a BLAST score of 70 (or in some cases 90) or greater thatdid not encode known proteins were clustered and assembled into aconsensus DNA sequence with the program “phrap” (Phil Green, Universityof Washington, Seattle, Wash.). The consensus sequence obtainedtherefrom is herein designated DNA56029.

In light of the sequence homology between the DNA56029 sequence and anEST sequence contained within the Incyte EST clone no. 2186536, theIncyte EST clone no. 2186536 was purchased and the cDNA insert wasobtained and sequenced. The sequence of this cDNA insert is shown inFIG. 9 and is herein designated as DNA59847-2510.

Clone DNA59847-2510 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 17-19 andending at the stop codon at nucleotide positions 1328-1330 (FIG. 9). Thepredicted polypeptide precursor is 437 amino acids long (FIG. 10). Thefull-length PRO1863 protein shown in FIG. 10 has an estimated molecularweight of about 46,363 daltons and a pI of about 6.22. Analysis of thefull-length PRO1863 sequence shown in FIG. 10 (SEQ ID NO:16) evidencesthe presence of the following: a signal peptide from about amino acid 1to about amino acid 15, a transmembrane domain from about amino acid 243to about amino acid 260, potential N-glycosylation sites from aboutamino acid 46 to about amino acid 49, from about amino acid 189 to aboutamino acid 192 and from about amino acid 382 to about amino acid 385,glycosaminoglycan attachment sites from about amino acid 51 to aboutamino acid 54 and from about amino acid 359 to about amino acid 362 andpotential N-myristolation sites from about amino acid 54 to about aminoacid 59, from about amino acid 75 to about amino acid 80, from aboutamino acid 141 to about amino acid 146, from about amino acid 154 toabout amino acid 159, from about amino acid 168 to about amino acid 173,from about amino acid 169 to about amino acid 174, from about amino acid198 to about amino acid 203, from about amino acid 254 to about aminoacid 259, from about amino acid 261 to about amino acid 266, from aboutamino acid 269 to about amino acid 274, from about amino acid 284 toabout amino acid 289, from about amino acid 333 to about amino acid 338,from about amino acid 347 to about amino acid 352, from about amino acid360 to about amino acid 365, from about amino acid 361 to about aminoacid 366, from about amino acid 388 to about amino acid 393, from aboutamino acid 408 to about amino acid 413 and from about amino acid 419 toabout amino acid 424. Clone DNA59847-2510 has been deposited with ATCCon Jan. 12, 1999 and is assigned ATCC deposit no. 203576.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 10 (SEQ ID NO:16), evidenced homology between the PRO1863amino acid sequence and the following Dayhoff sequences: AF041083_(—)1,P_W26579, HSA223603_(—)1, MMU97068, RNMAGPIAN_(—)1, CAHX_FLABR, S61882,AB007899_(—)1, CAH1_FLALI and P_W13386.

Example 9 Isolation of cDNA clones Encoding Human PRO1917

Use of the signal sequence algorithm described in Example 3 aboveallowed identification of an EST cluster sequence from the LIFESEQ®database, designated EST cluster no. 85496. This EST cluster sequencewas then compared to a the EST databases listed above to identifyexisting homologies. The homology search was performed using thecomputer program BLAST or BLAST2 (Altshul et al., Methods in Enzymology266:460-480 (1996)). Those comparisons resulting in a BLAST score of 70(or in some cases 90) or greater that did not encode known proteins wereclustered and assembled into a consensus DNA sequence with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.). Theconsensus sequence obtained therefrom is herein designated DNA56415.

In light of the sequence homology between the DNA56415 sequence and anEST sequence contained within EST no. 3255033, the EST clone, whichderived from an ovarian tumor library, was purchased and the cDNA insertwas obtained and sequenced. The sequence of this cDNA insert is shown inFIG. 11 and is herein designated as DNA76400-2528.

The full length clone shown in FIG. 11 contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 6-9 and ending at the stop codon found at nucleotide positions1467-1469 (FIG. 11; SEQ ID NO:17). The predicted polypeptide precursor(FIG. 12, SEQ ID NO:18) is 487 amino acids long. PRO1917 has acalculated molecular weight of approximately 55,051 daltons and anestimated pI of approximately 8.14. Additional features include: asignal peptide at about amino acids 1-30; potential N-glycosylationsites at about amino acids 242-245 and 481484, protein kinase Cphosphorylation sites at about amino acids 95-97, 182-184, and 427-429;N-myristoylation sites at about amino acids 107-112, 113-118, 117-122,118-123, and 128-133; and an endoplasmic reticulum targeting sequence atabout amino acids 484-487.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 12 (SEQ ID NO:18), revealed significant homology betweenthe PRO1917 amino acid sequence and Dayhoff sequence AF012714_(—)1.Significant homology was also revealed between the PRO1917 amino acidsequence and the sequence of a chondrocyte protein, designated“P_W52286” on the Dayhoff database, which has been reported to beinvolved in the transition of chondrocytes from proliferate tohypertrophic states (International Patent Application Publication No.WO9801468-A1). Homology was also revealed between the PRO1917 amino acidsequence and the following additional Dayhoff sequences: P_W52286,GGU59420_(—)1, P_R25597, PPA3_YEAST, PPA1_SCHPO, PPA2_SCHPO,A46783_(—)1, DMC165H7_(—)1, and AST8_DROME.

Clone DNA76400-2528 was deposited with the ATCC on Jan. 12, 1999, and isassigned ATCC deposit no. 203573.

Example 10 Isolation of cDNA clones Encoding Human PRO1868

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA49803. Based up an observed homology between theDNA49803 consensus sequence and an EST sequence contained within theIncyte EST clone no. 2994689, Incyte EST clone no. 2994689 was purchasedand its insert obtained and sequenced. The sequence of that insert isshown in FIG. 13 and is herein designated DNA77624-2515.

The entire nucleotide sequence of DNA77624-2515 is shown in FIG. 13 (SEQID NO:19). Clone DNA77624-2515 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 51-53and ending at the stop codon at nucleotide positions 981-983 (FIG. 13).The predicted polypeptide precursor is 310 amino acids long (FIG. 14).The full-length PRO1868 protein shown in FIG. 14 has an estimatedmolecular weight of about 35,020 daltons and a pI of about 7.90.Analysis of the full-length PRO1868 sequence shown in FIG. 14 (SEQ IDNO:20) evidences the presence of the following: a signal peptide fromabout amino acid 1 to about amino acid 30, a transmembrane domain fromabout amino acid 243 to about amino acid 263, potential N-glycosylationsites from about amino acid 104 to about amino acid 107 and from aboutamino acid 192 to about amino acid 195, a cAMP- and cGMP-dependentprotein kinase phosphorylation site from about amino acid 107 to aboutamino acid 110, casein kinase II phosphorylation sites from about aminoacid 106 to about amino acid 109 and from about amino acid 296 to aboutamino acid 299, a tyrosine kinase phosphorylation site from about aminoacid 69 to about amino acid 77 and potential N-myristolation sites fromabout amino acid 26 to about amino acid 31, from about amino acid 215 toabout amino acid 220, from about amino acid 226 to about amino acid 231,from about amino acid 243 to about amino acid 248, from about amino acid244 to about amino acid 249 and from about amino acid 262 to about aminoacid 267. Clone DNA77624-2515 has been deposited with ATCC on Dec. 22,1998 and is assigned ATCC deposit no. 203553.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 14 (SEQ ID NO:20), evidenced significant homology betweenthe PRO1868 amino acid sequence and the following Dayhoff sequences:HGS_RC75, P_W61379, A33_HUMAN, P_W14146, P_W14158, AMAL_DROME, P_R77437,I38346, NCM2_HUMAN and PTPD_HUMAN.

Example 11 Isolation of cDNA clones Encoding Human PRO3434

Use of the signal sequence algorithm described in Example 3 aboveallowed identification of an EST cluster sequence from the Incytedatabase. This EST cluster sequence was then compared to a variety ofexpressed sequence tag (EST) databases which included public ESTdatabases (e.g., GenBank) and a proprietary EST DNA database (Lifeseq®,Incyte Pharmaceuticals, Palo Alto, Calif.) to identify existinghomologies. The homology search was performed using the computer programBLAST or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480(1996)). Those comparisons resulting in a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into a consensus DNA sequence with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.). The consensussequence obtained therefrom is herein designated DNA56009.

In light of the sequence homology between the DNA56009 sequence and anEST sequence contained within the Incyte EST clone no 3327089, theIncyte EST clone no. 3327089 was purchased and the cDNA insert wasobtained and sequenced. The sequence of this cDNA insert is shown inFIG. 15 and is herein designated as DNA77631-2537.

Clone DNA77631-2537 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 46-48 andending at the stop codon at nucleotide positions 3133-3135 (FIG. 15).The predicted polypeptide precursor is 1029 amino acids long (FIG. 16).The full-length PRO3434 protein shown in FIG. 16 has an estimatedmolecular weight of about 114,213 daltons and a pI of about 6.42.Analysis of the full-length PRO3434 sequence shown in FIG. 16 (SEQ IDNO:22) evidences the presence of very important polypeptide domains asshown in FIG. 16. Clone DNA77631-2537 has been deposited with ATCC onFeb. 9, 1999 and is assigned ATCC deposit no. 203651.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 16 (SEQ ID NO:22), evidenced homology between the PRO3434amino acid sequence and the following Dayhoff sequences: VATX_YEAST,P_R51171, POLS_IBDVP, IBDVORF_(—)2. JC5043, IBDVPIV_(—)1, VE7_HPV11,GEN14220, MUTS_THETH and COAC_CHICK.

Example 12 Isolation of cDNA clones Encoding Human PRO1927

Use of the signal sequence algorithm described in Example 3 aboveallowed identification of an EST cluster sequence from the LIFESEQ®database, designated EST Cluster No. 1913. This EST cluster sequence wasthen compared to a variety of expressed sequence tag (EST) databaseswhich included the databases listed above, including an additionalproprietary EST DNA database (Genentech, South San Francisco, Calif.) toidentify existing homologies. The homology search was performed usingthe computer program BLAST or BLAST2 (Altshul et al., Methods inEnzymology 266:460-480 (1996)). Those comparisons resulting in a BLASTscore of 70 (or in some cases 90) or greater that did not encode knownproteins were clustered and assembled into a consensus DNA sequence withthe program “phrap” (Phil Green, University of Washington, Seattle,Wash.). The consensus sequence obtained therefrom is herein designatedDNA73896.

In light of the sequence homology between the DNA73896 sequence and anEST sequence contained within EST no. 3326981H1, EST clone no.3326981H1, which was obtained from a library constructed from RNAisolated from aortic tissue, was purchased and the cDNA insert wasobtained and sequenced. The sequence of this cDNA insert is shown inFIG. 17 and is herein designated as “DNA82307-2531”.

The full length clone shown in FIG. 17 contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 51-53 and ending at the stop codon found at nucleotidepositions 1695-1697 (FIG. 17; SEQ ID NO:23). The predicted polypeptideprecursor (FIG. 18, SEQ ID NO:24) is 548 amino acids long. PRO1927 has acalculated molecular weight of approximately 63,198 daltons and anestimated pI of approximately 8.10. Additional features include: asignal peptide at about amino acids 1-23; a putative transmembranedomain at about amino acids 6-25; potential N-glycosylation sites atabout amino acids 5-8, 87-90, 103-106, and 465469; potentialN-myristoylation sites at about amino acids 6-11, 136-141, 370-375, and509-514.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 18 (SEQ ID NO:24), revealed significant homology betweenthe PRO1927 amino acid sequence and Dayhoff sequence AB000628_(—)1.Homology was also revealed between the PRO1927 amino acid sequence andthe following additional Dayhoff sequences: HGS_A251, HGS_A197,CELC50H11_(—)2, CPXM_BACSU, VF03_VACCC, VF03_VACCV, DYHA_CHLRE, C69084,and A64315.

Clone DNA82307-2531 was deposited with the ATCC on Dec. 15, 1998, and isassigned ATCC deposit no. 203537.

Example 13 Inhibitory Activity in Mixed Lymphocyte Reaction (MLR) Assay(Assay 67)

This example shows that one or more of the polypeptides of the inventionare active as inhibitors of the proliferation of stimulatedT-lymphocytes. Compounds which inhibit proliferation of lymphocytes areuseful therapeutically where suppression of an immune response isbeneficial.

The basic protocol for this assay is described in Current Protocols inImmunology, unit 3.12; edited by J E Coligan, A M Kruisbeek, D HMarglies, E M Shevach, W Strober, National Insitutes of Health,Published by John Wiley & Sons, Inc.

More specifically, in one assay variant, peripheral blood mononuclearcells (PBMC) are isolated from mammalian individuals, for example ahuman volunteer, by leukopheresis (one donor will supply stimulatorPBMCs, the other donor will supply responder PBMCs). If desired, thecells are frozen in fetal bovine serum and DMSO after isolation. Frozencells may be thawed overnight in assay media (37° C., 5% CO₂) and thenwashed and resuspended to 3×10⁶ cells/ml of assay media (RPMI; 10% fetalbovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1%non-essential amino acids, 1% pyruvate). The stimulator PBMCs areprepared by irradiating the cells (about 3000 Rads).

The assay is prepared by plating in triplicate wells a mixture of:

-   -   100:1 of test sample diluted to 1% or to 0.1%,    -   50:1 of irradiated stimulator cells, and    -   50:1 of responder PBMC cells.        100 microliters of cell culture media or 100 microliter of        CD4-IgG is used as the control. The wells are then incubated at        37° C., 5% CO₂ for 4 days. On day 5, each well is pulsed with        tritiated thymidine (1.0 mC/well; Amersham). After 6 hours the        cells are washed 3 times and then the uptake of the label is        evaluated.

In another variant of this assay, PBMCs are isolated from the spleens ofBalb/c mice and C57B6 mice. The cells are teased from freshly harvestedspleens in assay media (RPMI; 10% fetal bovine serum, 1%penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential aminoacids, 1% pyruvate) and the PBMCs are isolated by overlaying these cellsover Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20minutes, collecting and washing the mononuclear cell layer in assaymedia and resuspending the cells to 1×10⁷ cells/ml of assay media. Theassay is then conducted as described above.

Any decreases below control is considered to be a positive result for aninhibitory compound, with decreases of less than or equal to 80% beingpreferred. However, any value less than control indicates an inhibitoryeffect for the test protein.

The following polypeptides tested positive in this assay: PRO1917 andPRO1868.

Example 14 Skin Vascular Permeability Assay (Assay 64)

This assay shows that certain polypeptides of the invention stimulate animmune response and induce inflammation by inducing mononuclear cell,eosinophil and PMN infiltration at the site of injection of the animal.Compounds which stimulate an immune response are useful therapeuticallywhere stimulation of an immune response is beneficial. This skinvascular permeability assay is conducted as follows. Hairless guineapigs weighing 350 grams or more are anesthetized with ketamine (75-80mg/Kg) and 5 mg/Kg xylazine intramuscularly (IM). A sample of purifiedpolypeptide of the invention or a conditioned media test sample isinjected intradermally onto the backs of the test animals with 100 μlper injection site. It is possible to have about 10-30, preferably about16-24, injection sites per animal. One μl of Evans blue dye (1% inphysiologic buffered saline) is injected intracardially. Blemishes atthe injection sites are then measured (mm diameter) at 1 hr and 6 hrpost injection. Animals were sacrificed at 6 hrs after injection. Eachskin injection site is biopsied and fixed in formalin. The skins arethen prepared for histopathologic evaluation. Each site is evaluated forinflammatory cell infiltration into the skin. Sites with visibleinflammatory cell inflammation are scored as positive. Inflammatorycells may be neutrophilic, eosinophilic, monocytic or lymphocytic. Atleast a minimal perivascular infiltrate at the injection site is scoredas positve, no infiltrate at the site of injection is scored asnegative.

The following polypeptides tested positive in this assay: PRO1434.

Example 15 Proliferation of Rat Utricular Supporting Cells (Assay 54)

This assay shows that certain polypeptides of the invention act aspotent mitogens for inner ear supporting cells which are auditory haircell progenitors and, therefore, are useful for inducing theregeneration of auditory hair cells and treating hearing loss inmammals. The assay is performed as follows. Rat UEC-4 utricularepithelial cells are aliquoted into 96 well plates with a density of3000 cells/well in 200 μl of serum-containing medium at 33° C. The cellsare cultured overnight and are then switched to serum-free medium at 37°C. Various dilutions of PRO polypeptides (or nothing for a control) arethen added to the cultures and the cells are incubated for 24 hours.After the 24 hour incubation, ³H-thymidine (1 μCi/well) is added and thecells are then cultured for an additional 24 hours. The cultures arethen washed to remove unincorporated radiolabel, the cells harvested andCpm per well determined. Cpm of at least 30% or greater in the PROpolypeptide treated cultures as compared to the control cultures isconsidered a positive in the assay.

The following polypeptides tested positive in this assay: PRO982.

Example 16 Gene Amplification

This example shows that the PRO1800-, PRO539-, PRO3434- andPRO1927-encoding genes are amplified in the genome of certain humanlung, colon and/or breast cancers and/or cell lines. Amplification isassociated with overexpression of the gene product, indicating that thepolypeptides are useful targets for therapeutic intervention in certaincancers such as colon, lung, breast and other cancers and diagnosticdetermination of the presence of those cancers. Therapeutic agents maytake the form of antagonists of PRO1800, PRO539, PRO3434 or PRO1927polypeptide, for example, murine-human chimeric, humanized or humanantibodies against a PRO1800, PRO539, PRO3434 or PRO1927 polypeptide.

The starting material for the screen was genomic DNA isolated from avariety cancers. The DNA is quantitated precisely, e.g.,fluorometrically. As a negative control, DNA was isolated from the cellsof ten normal healthy individuals which was pooled and used as assaycontrols for the gene copy in healthy individuals (not shown). The 5′nuclease assay (for example, TaqMan™) and real-time quantitative PCR(for example, ABI Prizm 7700 Sequence Detection System™ (Perkin Elmer,Applied Biosystems Division, Foster City, Calif.)), were used to findgenes potentially amplified in certain cancers. The results were used todetermine whether the DNA encoding PRO1800, PRO539, PRO3434 or PRO1927is over-represented in any of the primary lung or colon cancers orcancer cell lines or breast cancer cell lines that were screened. Theprimary lung cancers were obtained from individuals with tumors of thetype and stage as indicated in Table 7. An explanation of theabbreviations used for the designation of the primary tumors listed inTable 7 and the primary tumors and cell lines referred to throughoutthis example are given below.

The results of the TaqMan™ are reported in delta (Δ) Ct units. One unitcorresponds to 1 PCR cycle or approximately a 2-fold amplificationrelative to normal, two units corresponds to 4-fold, 3 units to 8-foldamplification and so on. Quantitation was obtained using primers and aTaqMan™ fluorescent probe derived from the PRO1800-, PRO539-, PRO3434-or PRO1927-encoding gene. Regions of PRO1800, PRO539, PRO3434 or PRO1927which are most likely to contain unique nucleic acid sequences and whichare least likely to have spliced out introns are preferred for theprimer and probe derivation, e.g., 3′-untranslated regions. Thesequences for the primers and probes (forward, reverse and probe) usedfor the PRO1800, PRO539, PRO3434 or PRO1927 gene amplification analysiswere as follows:

PRO1800 (DNA35672-2508) forward 5′-ACTCGGGATTCCTGCTGTT-3′ (SEQ ID NO:27)probe 5′-AGGCCTTTACCCAAGGCCACAAC-3′ (SEQ ID NO:28) reverse5′-GGCCTGTCCTGTGTTCTCA-3′ (SEQ ID NO:29) PRO539 (DNA47465-1561) forward5′-TCCCACCACTTACTTCCATGAA-3′ (SEQ ID NO:30) probe5′-CTGTGGTACCCAATTGCCGCCTTGT-3′ (SEQ ID NO:31) reverse5′-ATTGTCCTGAGATTCGAGCAAGA-3′ (SEQ ID NO :32) PRO3434 (DNA77631-2537)forward 5′-GTCCAGCAAGCCCTCATT-3′ (SEQ ID NO:33) probe5′-CTTCTGGGCCACAGCCCTGC-3′ (SEQ ID NO:34) reverse5′-CAGTTCAGGTCGTTTCATTCA-3′ (SEQ ID NO:35) PRO1927 (DNA82307-2531)forward 5′-CCAGTCAGGCCGTTTTAGA-3′ (SEQ ID NO:36) probe5′-CGGGCGCCCAAGTAAAAGCTC-3′ (SEQ ID NO:37) reverse5′-CATAAAGTAGTATATGCATTCCAGTGTT-3′ (SEQ ID NO:38)

The 5′ nuclease assay reaction is a fluorescent PCR-based techniquewhich makes use of the 5′ exonuclease activity of Taq DNA polymeraseenzyme to monitor amplification in real time. Two oligonucleotideprimers are used to generate an amplicon typical of a PCR reaction. Athird oligonucleotide, or probe, is designed to detect nucleotidesequence located between the two PCR primers. The probe isnon-extendible by Taq DNA polymerase enzyme, and is labeled with areporter fluorescent dye and a quencher fluorescent dye. Anylaser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

The 5′ nuclease procedure is run on a real-time quantitative PCR devicesuch as the ABI Prism 7700TM Sequence Detection. The system consists ofa thermocycler, laser, charge-coupled device (CCD) camera and computer.The system amplifies samples in a 96-well format on a thermocycler.During amplification, laser-induced fluorescent signal is collected inreal-time through fiber optics cables for all 96 wells, and detected atthe CCD. The system includes software for running the instrument and foranalyzing the data.

5′ Nuclease assay data are initially expressed as Ct, or the thresholdcycle. This is defined as the cycle at which the reporter signalaccumulates above the background level of fluorescence. The ΔCt valuesare used as quantitative measurement of the relative number of startingcopies of a particular target sequence in a nucleic acid sample whencomparing cancer DNA results to normal human DNA results.

Table 7 describes the stage, T stage and N stage of various primarytumors which were used to screen the PRO1800, PRO539, PRO3434 andPRO1927 compounds of the invention.

TABLE 7 Primary Lung and Colon Tumor Profiles Primary Tumor Stage StageOther Stage Dukes Stage T Stage N Stage Human lung tumor AdenoCa(SRCC724) [LT1] IIA T1 N1 Human lung tumor SqCCa (SRCC725) [LT1a] IIB T3N0 Human lung tumor AdenoCa (SRCC726) [LT2] IB T2 N0 Human lung tumorAdenoCa (SRCC727) [LT3] IIIA T1 N2 Human lung tumor AdenoCa (SRCC728)[LT4] IB T2 N0 Human lung tumor SqCCa (SRCC729) [LT6] IB T2 N0 Humanlung tumor Aden/SqCCa (SRCC730) [LT7] IA T1 N0 Human lung tumor AdenoCa(SRCC731) [LT9] IB T2 N0 Human lung tumor SqCCa (SRCC732) [LT10] IIB T2N1 Human lung tumor SqCCa (SRCC733) [LT11] IIA T1 N1 Human lung tumorAdenoCa (SRCC734) [LT12] IV T2 N0 Human lung tumor AdenoSqCCa (SRCC735)[LT13] IB T2 N0 Human lung tumor SqCCa (SRCC736) [LT15] IB T2 N0 Humanlung tumor SqCCa (SRCC737) [LT16] IB T2 N0 Human lung tumor SqCCa(SRCC738) [LT17] IIB T2 N1 Human lung tumor SqCCa (SRCC739) [LT18] IB T2N0 Human lung tumor SqCCa (SRCC740) [LT19] IB T2 N0 Human lung tumorLCCa (SRCC741) [LT21] IIB T3 N1 Human lung AdenoCa (SRCC811) [LT22] 1AT1 N0 Human colon AdenoCa (SRCC742) [CT2] M1 D pT4 N0 Human colonAdenoCa (SRCC743) [CT3] B pT3 N0 Human colon AdenoCa (SRCC744) [CT8] BT3 N0 Human colon AdenoCa (SRCC745) [CT10] A pT2 N0 Human colon AdenoCa(SRCC746) [CT12] MO, R1 B T3 N0 Human colon AdenoCa (SRCC747) [CT14]pMO, RO B pT3 pN0 Human colon AdenoCa (SRCC748) [CT15] M1, R2 D T4 N2Human colon AdenoCa (SRCC749) [CT16] pMO B pT3 pN0 Human colon AdenoCa(SRCC750) [CT17] C1 pT3 pN1 Human colon AdenoCa (SRCC751) [CT1] MO, R1 BpT3 N0 Human colon AdenoCa (SRCC752) [CT4] B pT3 M0 Human colon AdenoCa(SRCC753) [CT5] G2 C1 pT3 pN0 Human colon AdenoCa (SRCC754) [CT6] pMO,RO B pT3 pN0 Human colon AdenoCa (SRCC755) [CT7] G1 A pT2 pN0 Humancolon AdenoCa (SRCC756) [CT9] G3 D pT4 pN2 Human colon AdenoCa (SRCC757)[CT11] B T3 N0 Human colon AdenoCa (SRCC758) [CT18] MO, RO B pT3 pN0DNA Preparation:

DNA was prepared from cultured cell lines, primary tumors, normal humanblood. The isolation was performed using purification kit, buffer setand protease and all from Quiagen, according to the manufacturer'sinstructions and the description below.

Cell Culture Lysis:

Cells were washed and trypsinized at a concentration of 7.5×10⁸ per tipand pelleted by centrifuging at 1000 rpm for 5 minutes at 4° C.,followed by washing again with ½ volume of PBS recentrifugation. Thepellets were washed a third time, the suspended cells collected andwashed 2× with PBS. The cells were then suspended into 10 ml PBS. BufferC1 was equilibrated at 4° C. Qiagen protease #19155 was diluted into6.25 ml cold ddH₂O to a final concentration of 20 mg/ml and equilibratedat 4° C. 10 ml of G2 Buffer was prepared by diluting Qiagen RNAse Astock (100 mg/ml) to a final concentration of 200 μg/ml.

Buffer C1 (10 ml, 4° C.) and ddH2O (40 ml, 4° C.) were then added to the10 ml of cell suspension, mixed by inverting and incubated on ice for 10minutes. The cell nuclei were pelleted by centrifuging in a Beckmanswinging bucket rotor at 2500 rpm at 4° C. for 15 minutes. Thesupernatant was discarded and the nuclei were suspended with a vortexinto 2 ml Buffer C1 (at 4° C.) and 6 ml ddH₂O, followed by a second 4°C. centrifugation at 2500 rpm for 15 minutes. The nuclei were thenresuspended into the residual buffer using 200 μl per tip. G2 buffer (10ml) was added to the suspended nuclei while gentle vortexing wasapplied. Upon completion of buffer addition, vigorous vortexing wasapplied for 30 seconds. Quiagen protease (200 μl, prepared as indicatedabove) was added and incubated at 50° C. for 60 minutes. The incubationand centrifugation was repeated until the lysates were clear (e.g.,incubating additional 30-60 minutes, pelleting at 3000× g for 10 min.,4° C.).

Solid Human Tumor Sample Preparation and Lysis:

Tumor samples were weighed and placed into 50 ml conical tubes and heldon ice. Processing was limited to no more than 250 mg tissue perpreparation (1 tip/preparation). The protease solution was freshlyprepared by diluting into 6.25 ml cold ddH₂O to a final concentration of20 mg/ml and stored at 4° C. G2 buffer (20 ml) was prepared by dilutingDNAse A to a final concentration of 200 mg/ml (from 100 mg/ml stock).The tumor tissue was homogenated in 19 ml G2 buffer for 60 seconds usingthe large tip of the polytron in a laminar-flow TC hood in order toavoid inhalation of aerosols, and held at room temperature. Betweensamples, the polytron was cleaned by spinning at 2×30 seconds each in 2LddH₂O, followed by G2 buffer (50 ml). If tissue was still present on thegenerator tip, the apparatus was disassembled and cleaned.

Quiagen protease (prepared as indicated above, 1.0 ml) was added,followed by vortexing and incubation at 50° C. for 3 hours. Theincubation and centrifugation was repeated until the lysates were clear(e.g., incubating additional 30-60 minutes, pelleting at 3000× g for 10min., 4° C.).

Human Blood Preparation and Lysis:

Blood was drawn from healthy volunteers using standard infectious agentprotocols and citrated into 10 ml samples per tip. Quiagen protease wasfreshly prepared by dilution into 6.25 ml cold ddH₂O to a finalconcentration of 20 mg/ml and stored at 4° C. G2 buffer was prepared bydiluting RNAse A to a final concentration of 200 μg/ml from 100 mg/mlstock. The blood (10 ml) was placed into a 50 ml conical tube and 10 mlC1 buffer and 30 ml ddH₂O (both previously equilibrated to 4° C.) wereadded, and the components mixed by inverting and held on ice for 10minutes. The nuclei were pelleted with a Beckman swinging bucket rotorat 2500 rpm, 4° C. for 15 minutes and the supernatant discarded. With avortex, the nuclei were suspended into 2 ml C1 buffer (4° C.) and 6 mlddH₂O (4° C.). Vortexing was repeated until the pellet was white. Thenuclei were then suspended into the residual buffer using a 200 μl tip.G2 buffer (10 ml) were added to the suspended nuclei while gentlyvortexing, followed by vigorous vortexing for 30 seconds. Quiagenprotease was added (200 μl) and incubated at 50° C. for 60 minutes. Theincubation and centrifugation was repeated until the lysates were clear(e.g., incubating additional 30-60 minutes, pelleting at 3000× g for 10min., 4° C.).

Purification of Cleared Lysates:

(1) Isolation of Genomic DNA:

Genomic DNA was equilibrated (1 sample per maxi tip preparation) with 10ml QBT buffer. QF elution buffer was equilibrated at 50° C. The sampleswere vortexed for 30 seconds, then loaded onto equilibrated tips anddrained by gravity. The tips were washed with 2×15 ml QC buffer. The DNAwas eluted into 30 ml silanized, autoclaved 30 ml Corex tubes with 15 mlQF buffer (50° C.). Isopropanol (10.5 ml) was added to each sample, thetubes covered with parafin and mixed by repeated inversion until the DNAprecipitated. Samples were pelleted by centrifugation in the SS-34 rotorat 15,000 rpm for 10 minutes at 4° C. The pellet location was marked,the supernatant discarded, and 10 ml 70% ethanol (4° C.) was added.Samples were pelleted again by centrifugation on the SS-34 rotor at10,000 rpm for 10 minutes at 4° C. The pellet location was marked andthe supernatant discarded. The tubes were then placed on their side in adrying rack and dried 10 minutes at 37° C., taking care not to overdrythe samples.

After drying, the pellets were dissolved into 1.0 ml TE (pH 8.5) andplaced at 50° C. for 1-2 hours. Samples were held overnight at 4° C. asdissolution continued. The DNA solution was then transferred to 1.5 mltubes with a 26 gauge needle on a tuberculin syringe. The transfer wasrepeated 5× in order to shear the DNA. Samples were then placed at 50°C. for 1-2 hours.

(2) Quantitation of Genomic DNA and Preparation for Gene AmplificationAssay:

The DNA levels in each tube were quantified by standard A₂₆₀, A₂₈₀spectrophotometry on a 1:20 dilution (5 μl DNA +95 μl ddH₂O) using the0.1 ml quartz cuvetts in the Beckman DU640 spectrophotometer. A₂₆₀/A₂₈₀ratios were in the range of 1.8-1.9. Each DNA samples was then dilutedfurther to approximately 200 ng/ml in TE (pH 8.5). If the originalmaterial was highly concentrated (about 700 ng/μl), the material wasplaced at 50° C. for several hours until resuspended.

Fluorometric DNA quantitation was then performed on the diluted material(20-600 ng/ml) using the manufacturer's guidelines as modified below.This was accomplished by allowing a Hoeffer DyNA Quant 200 fluorometerto warm-up for about 15 minutes. The Hoechst dye working solution(#H33258, 10 μl, prepared within 12 hours of use) was diluted into 100ml 1× TNE buffer. A 2 ml cuvette was filled with the fluorometersolution, placed into the machine, and the machine was zeroed. pGEM3Zf(+) (2 μl, lot #360851026) was added to 2 ml of fluorometer solutionand calibrated at 200 units. An additional 2 μl of pGEM 3Zf(+) DNA wasthen tested and the reading confirmed at 400+/−10 units. Each sample wasthen read at least in triplicate. When 3 samples were found to be within10% of each other, their average was taken and this value was used asthe quantification value.

The fluorometricly determined concentration was then used to dilute eachsample to 10 ng/μl in ddH₂O. This was done simultaneously on alltemplate samples for a single TaqMan plate assay, and with enoughmaterial to run 500-1000 assays. The samples were tested in triplicatewith Taqman™ primers and probe both B-actin and GAPDH on a single platewith normal human DNA and no-template controls. The diluted samples wereused provided that the CT value of normal human DNA subtracted from testDNA was +/−1 Ct. The diluted, lot-qualified genomic DNA was stored in1.0 ml aliquots at −80° C. Aliquots which were subsequently to be usedin the gene amplification assay were stored at 4° C. Each 1 ml aliquotis enough for 8-9 plates or 64 tests.

Gene Amplification Assay:

The PRO1800, PRO539, PRO3434 and PRO1927 compounds of the invention werescreened in the following primary tumors and the resulting ΔCt valuesgreater than or equal to 1.0 are reported in Table 8 below.

TABLE 8 (ΔCt values in lung and colon primary tumor models) PrimaryTumor PRO1800 PRO539 PRO3434 PRO1927 LT11 1.65, 1.59, 1.03 LT12 1.34,2.28, 2.03 1.25 LT13 1.27, 2.18 1.64, 1.08 5.24, 4.47 4.38, 4.80 LT151.70, 2.23, 1.93 1.78, 1.10 1.24 1.00 LT16 1.00, 1.05, 1.09 3.65, 3.192.73, 2.74 LT17 1.94, 1.63 1.94, 1.01 LT18 1.12 LT19 2.51, 2.18 1.16LT21 1.30 1.32 CT2 1.50 CT3 1.17 CT10 1.16 CT12 1.19 CT14 1.62 CT151.48, 1.08 1.03 1.19, 1.40 1.10, 1.30 CT5 1.10 CT11 1.20 1.12 Colo-3201.16 1.78, 1.76, 1.74 1.51 (colon tumor cell line) HF-00084 2.20 2.41(lung tumor cell line) HCT-116 2.15, 2.22 1.41, 1.47 (colon tumor cellline) HF-00129 1.00, 1.17, 4.64 2.31, 5.14 (lung tumor cell line) 1.112.40 SW-620 1.30 (colon tumor cell line) HT-29 1.64 (colon tumor cellline) SW-403 1.75 (colon tumor cell line) LS174T 1.42 (colon tumor cellline) HCC-2998 1.15 (colon tumor cell line) A549 1.51, 1.09 (lung tumorcell line) Calu-6 1.60, 1.22 (lung tumor cell line) H157 1.61 (lungtumor cell line) H441 1.07, 1.15 (lung tumor cell line) H460 1.01 (lungtumor cell line) SKMES1 1.02 (lung tumor cell line) H810 1.20, 1.54(lung tumor cell line)

Example 17 Induction of Pancreatic β-Cell Precursor Proliferation (Assay117)

This assay shows that certain polypeptides of the invention act toinduce an increase in the number of pancreatic β-cell precursor cellsand, therefore, are useful for treating various insulin deficient statesin mammals, including diabetes mellitus. The assay is performed asfollows. The assay uses a primary culture of mouse fetal pancreaticcells and the primary readout is an alteration in the expression ofmarkers that represent either β-cell precursors or mature β-cells.Marker expression is measured by real time quantitative PCR (RTQ-PCR);wherein the marker being evaluated is a transcription factor calledPdx1.

The pancreata are dissected from E14 embryos (CD1 mice). The pancreataare then digested with collagenase/dispase in F12/DMEM at 37° C. for 40to 60 minutes (collagenase/dispase, 1.37 mg/ml, Boehringer Mannheim,#1097113). The digestion is then neutralized with an equal volume of 5%BSA and the cells are washed once with RPMI 1640. At day 1, the cellsare seeded into 12-well tissue culture plates (pre-coated with laminin,20 μg/ml in PBS, Boehringer Mannheim, #124317). Cells from pancreatafrom 1-2 embryos are distributed per well. The culture medium for thisprimary cuture is 14F/1640. At day 2, the media is removed and theattached cells washed with RPMI/1640. Two mls of minimal media are addedin addition to the protein to be tested. At day 4, the media is removedand RNA prepared from the cells and marker expression analyzed by realtime quantitative RT-PCR. A protein is considered to be active in theassay if it increases the expression of the relevant β-cell marker ascompared to untreated controls.

14F/1640 is RPMI1640 (Gibco) Plus the Following:

-   -   group A 1:1000    -   group B 1:1000    -   recombinant human insulin 10 μg/ml    -   Aprotinin (50 μg/ml) 1:2000 (Boehringer manheim #981532)    -   Bovine pituitary extract (BPE) 60 μg/ml    -   Gentamycin 100 ng/ml        Group A: (in 10 ml PBS)    -   Transferrin, 100 mg (Sigma T2252)    -   Epidermal Growth Factor, 100 μg (BRL 100004)    -   Triiodothyronine, 10 μl of 5×10⁻⁶ M (Sigma T5516)    -   Ethanolamine, 100 μl of 10⁻¹ M (Sigma E0135)    -   Phosphoethalamine, 100 μl of 10⁻¹ M (Sigma P0503)    -   Selenium, 4 μl of 10⁻¹ M (Aesar #12574)        Group C: (in 10 ml 100% Ethanol)    -   Hydrocortisone, 2 μl of 5×10⁻³ M (Sigma #H0135)    -   Progesterone, 100 μl of 1×10⁻³ M (Sigma #P6149)    -   Forskolin, 500 μl of 20 mM (Calbiochem #344270)        Minimal Media:    -   RPMI 1640 plus transferrin (10 μg/ml), insulin (1 μg/ml),        gentamycin (100 ng/ml), aprotinin (50 μg/ml) and BPE (15 μg/ml).        Defined Media:

RPMI 1640 plus transferrin (10 μg/ml), insulin (1 μg/ml), gentamycin(100 ng/ml) and aprotinin (50 μg/ml).

The following polypeptide was positive in this assay: PRO1868.

Example 18 Induction of Pancreatic β-Cell Precursor Differentiation(Assay 89)

This assay shows that certain polypeptides of the invention act toinduce differentiation of pancreatic β-cell precursor cells into maturepancreatic β-cells and, therefore, are useful for treating variousinsulin deficient states in mammals, including diabetes mellitus. Theassay is performed as follows. The assay uses a primary culture of mousefetal pancreatic cells and the primary readout is an alteration in theexpression of markers that represent either β-cell precursors or matureβ-cells. Marker expression is measured by real time quantitative PCR(RTQ-PCR); wherein the marker being evaluated is insulin.

The pancreata are dissected from E14 embryos (CD1 mice). The pancreataare then digested with collagenase/dispase in F12/DMEM at 37° C. for 40to 60 minutes (collagenase/dispase, 1.37 mg/ml, Boehringer Mannheim,#1097113). The digestion is then neutralized with an equal volume of 5%BSA and the cells are washed once with RPM11640. At day 1, the cells areseeded into 12-well tissue culture plates (pre-coated with laminin, 20μg/ml in PBS, Boehringer Mannheim, #124317). Cells from pancreata from1-2 embryos are distributed per well. The culture medium for thisprimary cuture is 14F/1640. At day 2, the media is removed and theattached cells washed with RPMI/1640. Two mls of minimal media are addedin addition to the protein to be tested. At day 4, the media is removedand RNA prepared from the cells and marker expression analyzed by realtime quantitative RT-PCR. A protein is considered to be active in theassay if it increases the expression of the relevant β-cell marker ascompared to untreated controls.

14F/1640 is RPMI1640 (Gibco) Plus the Following:

-   -   group A 1:1000    -   group B 1:1000    -   recombinant human insulin 10 μg/ml    -   Aprotinin (50 μg/ml) 1:2000 (Boehringer manheim #981532)    -   Bovine pituitary extract (BPE) 60 μg/ml    -   Gentamycin 100 ng/ml        Group A: (in 10 ml PBS)    -   Transferrin, 100 mg (Sigma T2252)    -   Epidermal Growth Factor, 100 μg (BRL 100004)    -   Triiodothyronine, 10 μl of 5×10⁻⁶ M (Sigma T5516)    -   Ethanolamine, 100 μl of 10⁻¹ M (Sigma E0135)    -   Phosphoethalamine, 100 μl of 10⁻¹ M (Sigma P0503)    -   Selenium, 4 μl of 10⁻¹ M (Aesar #12574)        Group C: (in 10 ml 100% Ethanol)    -   Hydrocortisone, 2 μl of 5×10⁻³ M (Sigma #H0135)    -   Progesterone, 100 μl of 1×10⁻³ M (Sigma #P6149)    -   Forskolin, 500 μl of 20 mM (Calbiochem #344270)        Minimal Media:    -   RPMI 1640 plus transferrin (10 μg/ml), insulin (1 μg/ml),        gentamycin (100 ng/ml), aprotinin (50 μg/ml) and BPE (15 μg/ml).        Defined Media:    -   RPMI 1640 plus transferrin (10 μg/ml), insulin (1 μg/ml),        gentamycin (100 ng/ml) and aprotinin (50 μg/ml).

The following polypeptide was positive in this assay: PRO1863.

Example 19 Mouse Kidney Mesangial Cell Proliferation Assay (Assay 92)

This assay shows that certain polypeptides of the invention act toinduce proliferation of mammalian kidney mesangial cells and, therefore,are useful for treating kidney disorders associated with decreasedmesangial cell function such as Berger disease or other nephropathiesassociated with Schönlein-Henoch purpura, celiac disease, dermatitisherpetiformis or Crohn disease. The assay is performed as follows. Onday one, mouse kidney mesangial cells are plated on a 96 well plate ingrowth media (3:1 mixture of Dulbecco's modified Eagle's medium andHam's F12 medium, 95% fetal bovine serum, 5% supplemented with 14 mMHEPES) and grown overnight. On day 2, PRO polypeptides are diluted at 2concentrations(1% and 0.1%) in serum-free medium and added to the cells.Control samples are serum-free medium alone. On day 4, 20 μl of the CellTiter 96 Aqueous one solution reagent (Progema) was added to each welland the colormetric reaction was allowed to proceed for 2 hours. Theabsorbance (OD) is then measured at 490 nm. A positive in the assay isanything that gives an absorbance reading which is at least 15% abovethe control reading.

The following polypeptide tested positive in this assay: PRO1917.

Example 20 Fibroblast (BHK-21) Proliferation (Assay 98)

This assay shows that certain polypeptides of the invention act toinduce proliferation of mammalian fibroblast cells in culture and,therefore, function as useful growth factors in mammalian systems. Theassay is performed as follows. BHK-21 fibroblast cells plated instandard growth medium at 2500 cells/well in a total volume of 100 μl .The PRO polypeptide, β-FGF (positive control) or nothing (negativecontrol) are then added to the wells in the presence of 1 μg/ml ofheparin for a total final volume of 200 μl. The cells are then incubatedat 37° C. for 6 to 7 days. After incubation, the media is removed, thecells are washed with PBS and then an acid phosphatase substratereaction mixture (100 μl/well) is added. The cells are then incubated at37° C. for 2 hours. 10 μl per well of 1N NaOH is then added to stop theacid phosphatase reaction. The plates are then read at OD 405 nm. Apositive in the assay is acid phosphatase activity which is at least 50%above the negative control.

The following polypeptide tested positive in this assay: PRO982.

Example 21 Chondrocyte Re-differentiation Assay (Assay 110)

This assay shows that certain polypeptides of the invention act toinduce redifferentiation of chondrocytes, therefore, are expected to beuseful for the treatment of various bone and/or cartilage disorders suchas, for example, sports injuries and arthritis. The assay is performedas follows. Porcine chondrocytes are isolated by overnight collagenasedigestion of articulary cartilage of metacarpophalangeal joints of 4-6month old female pigs. The isolated cells are then seeded at 25,000cells/cm² in Ham F-12 containing 10% FBS and 4 μg/ml gentamycin. Theculture media is changed every third day and the cells are then seededin 96 well plates at 5,000 cells/well in 100 μl of the same mediawithout serum and 100 μl of the test PRO polypeptide, 5 nM staurosporin(positive control) or medium alone (negative control) is added to give afinal volume of 200 μl/well. After 5 days of incubation at 37° C., apicture of each well is taken and the differentiation state of thechondrocytes is determined. A positive result in the assay occurs whenthe redifferentiation of the chondrocytes is determined to be moresimilar to the positive control than the negative control.

The following polypeptide tested positive in this assay: PRO1863.

Example 22 Use of PRO as a Hybridization Probe

The following method describes use of a nucleotide sequence encoding PROas a hybridization probe.

DNA comprising the coding sequence of full-length or mature PRO asdisclosed herein is employed as a probe to screen for homologous DNAs(such as those encoding naturally-occurring variants of PRO) in humantissue cDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled PRO-derived probe to the filters is performed in asolution of 50% formamide, 5× SSC, 0.1% SDS, 0.1% sodium pyrophosphate,50 mM sodium phosphate, pH 6.8, 2× Denhardt's solution, and 10% dextransulfate at 42° C. for 20 hours. Washing of the filters is performed inan aqueous solution of 0.1× SSC and 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence PRO can then be identified using standardtechniques known in the art.

Example 23 Expression of PRO in E. coli

This example illustrates preparation of an unglycosylated form of PRO byrecombinant expression in E. coli.

The DNA sequence encoding PRO is initially amplified using selected PCRprimers. The primers should contain restriction enzyme sites whichcorrespond to the restriction enzyme sites on the selected expressionvector. A variety of expression vectors may be employed. An example of asuitable vector is pBR322 (derived from E. coli; see Bolivar et al.,Gene 2.95 (1977)) which contains genes for ampicillin and tetracyclineresistance. The vector is digested with restriction enzyme anddephosphorylated. The PCR amplified sequences are then ligated into thevector. The vector will preferably include sequences which encode for anantibiotic resistance gene, a trp promoter, a polyhis leader (includingthe first six STII codons, polyhis sequence, and enterokinase cleavagesite), the PRO coding region, lambda transcriptional terminator, and anargU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

PRO may be expressed in E. coli in a poly-His tagged form, using thefollowing procedure. The DNA encoding PRO is initially amplified usingselected PCR primers. The primers will contain restriction enzyme siteswhich correspond to the restriction enzyme sites on the selectedexpression vector, and other useful sequences providing for efficientand reliable translation initiation, rapid purification on a metalchelation column, and proteolytic removal with enterokinase. ThePCR-amplified, poly-His tagged sequences are then ligated into anexpression vector, which is used to transform an E. coli host based onstrain 52 (W3110 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(lacIq).Transformants are first grown in LB containing 50 mg/ml carbenicillin at30° C. with shaking until an O.D.600of 3-5 is reached. Cultures are thendiluted 50-100 fold into CRAP media (prepared by mixing 3.57 g(NH₄)₂SO₄, 0.71 g sodium citrate.2H2O, 1.07 g KCl, 5.36 g Difco yeastextract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mMMPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grown forapproximately 20-30 hours at 30° C. with shaking. Samples are removed toverify expression by SDS-PAGE analysis, and the bulk culture iscentrifuged to pellet the cells. Cell pellets are frozen untilpurification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant is diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 ml QiagenNi-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

The proteins are refolded by diluting the sample slowly into freshlyprepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refoldingvolumes are chosen so that the final protein concentration is between 50to 100 micrograms/ml. The refolding solution is stirred gently at 4° C.for 12-36 hours. The refolding reaction is quenched by the addition ofTFA to a final concentration of 0.4% (pH of approximately 3). Beforefurther purification of the protein, the solution is filtered through a0.22 micron filter and acetonitrile is added to 2-10% finalconcentration. The refolded protein is chromatographed on a Poros R1/Hreversed phase column using a mobile buffer of 0.1% TFA with elutionwith a gradient of acetonitrile from 10 to 80%. Aliquots of fractionswith A280 absorbance are analyzed on SDS polyacrylamide gels andfractions containing homogeneous refolded protein are pooled. Generally,the properly refolded species of most proteins are eluted at the lowestconcentrations of acetonitrile since those species are the most compactwith their hydrophobic interiors shielded from interaction with thereversed phase resin. Aggregated species are usually eluted at higheracetonitrile concentrations. In addition to resolving misfolded forms ofproteins from the desired form, the reversed phase step also removesendotoxin from the samples.

Fractions containing the desired folded PRO polypeptide are pooled andthe acetonitrile removed using a gentle stream of nitrogen directed atthe solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14M sodium chloride and 4% mannitol by dialysis or by gel filtration usingG25 Superfine (Pharmacia) resins equilibrated in the formulation bufferand sterile filtered.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 24 Expression of PRO in Mammalian Cells

This example illustrates preparation of a potentially glycosylated formof PRO by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the PRO DNA is ligated into pRK5with selected restriction enzymes to allow insertion of the PRO DNAusing ligation methods such as described in Sambrook et al., supra. Theresulting vector is called pRK5-PRO.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μg pRK5-PRODNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappayaet al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl,0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added, dropwise, 500 μlof 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄, and a precipitateis allowed to form for 10 minutes at 25° C. The precipitate is suspendedand added to the 293 cells and allowed to settle for about four hours at37° C. The culture medium is aspirated off and 2 ml of 20% glycerol inPBS is added for 30 seconds. The 293 cells are then washed with serumfree medium, fresh medium is added and the cells are incubated for about5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of PRO polypeptide. The cultures containing transfected cellsmay undergo further incubation (in serum free medium) and the medium istested in selected bioassays.

In an alternative technique, PRO may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg pRK5-PRO DNA is added. Thecells are first concentrated from the spinner flask by centrifugationand washed with PBS. The DNA-dextran precipitate is incubated on thecell pellet for four hours. The cells are treated with 20% glycerol for90 seconds, washed with tissue culture medium, and re-introduced intothe spinner flask containing tissue culture medium, 5 μg/ml bovineinsulin and 0.1 μg/ml bovine transferrin. After about four days, theconditioned media is centrifuged and filtered to remove cells anddebris. The sample containing expressed PRO can then be concentrated andpurified by any selected method, such as dialysis and/or columnchromatography.

In another embodiment, PRO can be expressed in CHO cells. The pRK5-PROcan be transfected into CHO cells using known reagents such as CaPO₄ orDEAE-dextran. As described above, the cell cultures can be incubated,and the medium replaced with culture medium (alone) or medium containinga radiolabel such as ³⁵S-methionine. After determining the presence ofPRO polypeptide, the culture medium may be replaced with serum freemedium. Preferably, the cultures are incubated for about 6 days, andthen the conditioned medium is harvested. The medium containing theexpressed PRO can then be concentrated and purified by any selectedmethod.

Epitope-tagged PRO may also be expressed in host CHO cells. The PRO maybe subcloned out of the pRK5 vector. The subclone insert can undergo PCRto fuse in frame with a selected epitope tag such as a poly-his tag intoa Baculovirus expression vector. The poly-his tagged PRO insert can thenbe subcloned into a SV40 driven vector containing a selection markersuch as DHFR for selection of stable clones. Finally, the CHO cells canbe transfected (as described above) with the SV40 driven vector.Labeling may be performed, as described above, to verify expression. Theculture medium containing the expressed poly-His tagged PRO can then beconcentrated and purified by any selected method, such as byNi²⁺-chelate affinity chromatography.

PRO may also be expressed in CHO and/or COS cells by a transientexpression procedure or in CHO cells by another stable expressionprocedure.

Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins are fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or is a poly-His tagged form.

Following PCR amplification, the respective DNAs are subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are constructed to have compatiblerestriction sites 5′ and 3′ of the DNA of interest to allow theconvenient shuttling of cDNA's. The vector used expression in CHO cellsis as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Quiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁻⁷ cells are frozen in an ampule for furthergrowth and production as described below.

The ampules containing the plasmid DNA are thawed by placement intowater bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2-3 days,250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3 L production spinner isseeded at 1.2×10⁶ cells/mL. On day 0, the cell number pH ie determined.On day 1, the spinner is sampled and sparging with filtered air iscommenced. On day 2, the spinner is sampled, the temperature shifted to33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g.,35% polydimethylsiloxane emulsion, Dow Corning 365 Medical GradeEmulsion) taken. Throughout the production, the pH is adjusted asnecessary to keep it at around 7.2. After 10 days, or until theviability dropped below 70%, the cell culture is harvested bycentrifugation and filtering through a 0.22 μm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

For the poly-His tagged constructs, the proteins are purified using aNi-NTA column (Qiagen). Before purification, imidazole is added to theconditioned media to a concentration of 5 mM. The conditioned media ispumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5ml/min. at 4° C. After loading, the column is washed with additionalequilibration buffer and the protein eluted with equilibration buffercontaining 0.25 M imidazole. The highly purified protein is subsequentlydesalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column andstored at -80° C.

Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 25 Expression of PRO in Yeast

The following method describes recombinant expression of PRO in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO from the ADH2/GAPDH promoter. DNAencoding PRO and the promoter is inserted into suitable restrictionenzyme sites in the selected plasmid to direct intracellular expressionof PRO. For secretion, DNA encoding PRO can be cloned into the selectedplasmid, together with DNA encoding the ADH2/GAPDH promoter, a nativePRO signal peptide or other mammalian signal peptide, or, for example, ayeast alpha-factor or invertase secretory signal/leader sequence, andlinker sequences (if needed) for expression of PRO.

Yeast cells, such as yeast strain AB 110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant PRO can subsequently be isolated and purified by removingthe yeast cells from the fermentation medium by centrifugation and thenconcentrating the medium using selected cartridge filters. Theconcentrate containing PRO may further be purified using selected columnchromatography resins.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 26 Expression of PRO in Baculovirus-infected Insect Cells

The following method describes recombinant expression of PRO inBaculovirus-infected insect cells.

The sequence coding for PRO is fused upstream of an epitope tagcontained within a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thesequence encoding PRO or the desired portion of the coding sequence ofPRO such as the sequence encoding the extracellular domain of atransmembrane protein or the sequence encoding the mature protein if theprotein is extracellular is amplified by PCR with primers complementaryto the 5′ and 3′ regions. The 5′ primer may incorporate flanking(selected) restriction enzyme sites. The product is then digested withthose selected restriction enzymes and subcloned into the expressionvector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged PRO can then be purified, for example, byNi²⁺-chelate affinity chromatography as follows. Extracts are preparedfrom recombinant virus-infected Sf9 cells as described by Rupert et al.,Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspendedin sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl₂; 0.1 mM EDTA;10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 secondson ice. The sonicates are cleared by centrifugation, and the supernatantis diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni²⁺-NTAagarose column (commercially available from Qiagen) is prepared with abed volume of 5 mL, washed with 25 mL of water and equilibrated with 25mL of loading buffer. The filtered cell extract is loaded onto thecolumn at 0.5 mL per minute. The column is washed to baseline A₂₈₀ withloading buffer, at which point fraction collection is started. Next, thecolumn is washed with a secondary wash buffer (50 mM phosphate; 300 mMNaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.After reaching A₂₈₀ baseline again, the column is developed with a 0 to500 mM Imidazole gradient in the secondary wash buffer. One mL fractionsare collected and analyzed by SDS-PAGE and silver staining or Westernblot with Ni²⁺-NTA-conjugated to alkaline phosphatase (Qiagen).Fractions containing the eluted His₁₀-tagged PRO are pooled and dialyzedagainst loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) PRO can beperformed using known chromatography techniques, including for instance,Protein A or protein G column chromatography.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 27 Preparation of Antibodies that Bind PRO

This example illustrates preparation of monoclonal antibodies which canspecifically bind PRO.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified PRO, fusion proteins containing PRO, andcells expressing recombinant PRO on the cell surface. Selection of theimmunogen can be made by the skilled artisan without undueexperimentation.

Mice, such as Balb/c, are immunized with the PRO immunogen emulsified incomplete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-PRO antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO. Three to four days later, the mice are sacrificed andthe spleen cells are harvested. The spleen cells are then fused (using35% polyethylene glycol) to a selected murine myeloma cell line such asP3X63AgU. 1, available from ATCC, No. CRL 1597. The fusions generatehybridoma cells which can then be plated in 96 well tissue cultureplates containing HAT (hypoxanthine, aminopterin, and thymidine) mediumto inhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstPRO. Determination of “positive” hybridoma cells secreting the desiredmonoclonal antibodies against PRO is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-PROmonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 28 Purification of PRO Polypeptides Using Specific Antibodies

Native or recombinant PRO polypeptides may be purified by a variety ofstandard techniques in the art of protein purification. For example,pro-PRO polypeptide, mature PRO polypeptide, or pre-PRO polypeptide ispurified by immunoaffinity chromatography using antibodies specific forthe PRO polypeptide of interest. In general, an immunoaffinity column isconstructed by covalently coupling the anti-PRO polypeptide antibody toan activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such an immunoaffinity column is utilized in the purification of PROpolypeptide by preparing a fraction from cells containing PROpolypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble PRO polypeptidecontaining a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

A soluble PRO polypeptide-containing preparation is passed over theimmunoaffinity column, and the column is washed under conditions thatallow the preferential absorbance of PRO polypeptide (e.g., high ionicstrength buffers in the presence of detergent). Then, the column iseluted under conditions that disrupt antibody/PRO polypeptide binding(e.g., a low pH buffer such as approximately pH 2-3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), and PROpolypeptide is collected.

Example 29 Drug Screening

This invention is particularly useful for screening compounds by usingPRO polypeptides or binding fragment thereof in any of a variety of drugscreening techniques. The PRO polypeptide or fragment employed in such atest may either be free in solution, affixed to a solid support, borneon a cell surface, or located intracellularly. One method of drugscreening utilizes eukaryotic or prokaryotic host cells which are stablytransformed with recombinant nucleic acids expressing the PROpolypeptide or fragment. Drugs are screened against such transformedcells in competitive binding assays. Such cells, either in viable orfixed form, can be used for standard binding assays. One may measure,for example, the formation of complexes between PRO polypeptide or afragment and the agent being tested. Alternatively, one can examine thediminution in complex formation between the PRO polypeptide and itstarget cell or target receptors caused by the agent being tested.

Thus, the present invention provides methods of screening for drugs orany other agents which can affect a PRO polypeptide-associated diseaseor disorder. These methods comprise contacting such an agent with an PROpolypeptide or fragment thereof and assaying (I) for the presence of acomplex between the agent and the PRO polypeptide or fragment, or (ii)for the presence of a complex between the PRO polypeptide or fragmentand the cell, by methods well known in the art. In such competitivebinding assays, the PRO polypeptide or fragment is typically labeled.After suitable incubation, free PRO polypeptide or fragment is separatedfrom that present in bound form, and the amount of free or uncomplexedlabel is a measure of the ability of the particular agent to bind to PROpolypeptide or to interfere with the PRO polypeptide/cell complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to a polypeptide and isdescribed in detail in WO 84/03564, published on Sep. 13, 1984. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. As applied to a PRO polypeptide, the peptide test compounds arereacted with PRO polypeptide and washed. Bound PRO polypeptide isdetected by methods well known in the art. Purified PRO polypeptide canalso be coated directly onto plates for use in the aforementioned drugscreening techniques. In addition, non-neutralizing antibodies can beused to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding PROpolypeptide specifically compete with a test compound for binding to PROpolypeptide or fragments thereof. In this manner, the antibodies can beused to detect the presence of any peptide which shares one or moreantigenic determinants with PRO polypeptide.

Example 30 Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptide of interest (i.e., a PRO polypeptide) orof small molecules with which they interact, e.g., agonists,antagonists, or inhibitors. Any of these examples can be used to fashiondrugs which are more active or stable forms of the PRO polypeptide orwhich enhance or interfere with the function of the PRO polypeptide invivo (c.f., Hodgson, Bio/Technology, 9: 19-21 (1991)).

In one approach, the three-dimensional structure of the PRO polypeptide,or of an PRO polypeptide-inhibitor complex, is determined by x-raycrystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of the PROpolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of the PRO polypeptide may be gained by modelingbased on the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous PRO polypeptide-likemolecules or to identify efficient inhibitors. Useful examples ofrational drug design may include molecules which have improved activityor stability as shown by Braxton and Wells, Biochemistry. 31:7796-7801(1992) or which act as inhibitors, agonists, or antagonists of nativepeptides as shown by Athauda et al., J. Biochem., 113:742-746 (1993).

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amounts of the PROpolypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, knowledge of the PRO polypeptideamino acid sequence provided herein will provide guidance to thoseemploying computer modeling techniques in place of or in addition tox-ray crystallography.

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md., USA (ATCC):

Material ATCC Dep. No. Deposit Date DNA35672-2508 203538 Dec. 15, 1998DNA47465-1561 203661 Feb. 9, 1999 DNA57700-1408 203583 Jan. 12, 1999DNA68818-2536 203657 Feb. 9, 1999 DNA59847-2510 203576 Jan. 12, 1999DNA76400-2528 203573 Jan. 12, 1999 DNA77624-2515 203553 Dec. 22, 1998DNA77631-2537 203651 Feb. 9, 1999 DNA82307-2531 203537 Dec. 15, 1998

These deposit were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposits will be made availableby ATCC under the terms of the Budapest Treaty, and subject to anagreement between Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC § 122 and the Commissioner's rules pursuantthereto (including 37 CFR § 1.14 with particular reference to 886 OG638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. An isolated nucleic acid comprising: (a) a nucleic acid sequenceencoding the polypeptide of SEQ ID NO:7; (b) the nucleic acid sequenceof SEQ ID NO:6; (c) the full-length coding sequence of the nucleic acidsequence of SEQ ID NO:6; or (d) the full-length coding sequence of thecDNA deposited under ATCC accession number
 203661. 2. The isolatednucleic acid of claim 1 comprising a nucleic acid sequence encoding thepolypeptide of SEQ ID NO:7.
 3. The isolated nucleic acid of claim 1comprising the nucleic acid sequence of SEQ ID NO:6.
 4. The isolatednucleic acid of claim 1 comprising the full-length coding sequence ofthe nucleic acid sequence of SEQ ID NO:6.
 5. The isolated nucleic acidof claim 1 comprising the full-length coding sequence of the cDNAdeposited under ATCC accession number 203661.